For Research Use Only. Not for use in diagnostic procedures.

SuperScript® Plasmid System with Gateway® Technology for cDNA Synthesis and Cloning Catalog Number 18248-013, 109625011

Document Part Number 11108Publication Number MAN0000416Revision A.0

Table of Contents

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1. Notices to Customer ................................................................................ 1 1.1 Important Information ....................................................................................... 1 1.2 Precautions ....................................................................................................... 1 1.3 Limited Label Licenses ......................................................................................1 1.4 Trademarks ........................................................................................................2 1.5 Disclaimer...........................................................................................................2

2. Overview ....................................................................................................... 3 2.1 cDNA Libraries .................................................................................................. 3 2.2 mRNA Isolation ................................................................................................. 4 2.3 First Strand Synthesis ...................................................................................... 5 2.4 Second Strand Synthesis ................................................................................. 5 2.5 Constructing Directional Libraries .................................................................... 6 2.5.1 Introducing Asymmetry into cDNA ......................................................... 6 2.5.2 Maximizing the Ligation Efficiency of cDNA to the Vector by Adapter Addition ........................................................ 6 2.5.3 Size Fractionation of cDNA .................................................................... 7 2.5.4 Ligation of Size-Fractionated cDNA to the Plasmid Vector and Introduction of DNA into E. coli......................................... 8 2.6 Gateway® Cloning Technology ........................................................................ 8 2.7 Cloning Vectors ............................................................................................... 8 2.7.1 PlasmidpCMV•SPORT6....................................................................... 9 2.7.2 PlasmidpSPORT1 .............................................................................. 10 2.7.3 PlasmidpSPORT2 .............................................................................. 10 2.7.4 Plasmid pEXP-AD502 .......................................................................... 10

3. Methods ....................................................................................................... 13 3.1 Components ................................................................................................... 13 3.2 General Comments ........................................................................................ 13 3.2.1 mRNA Purification ................................................................................ 13 3.2.2 Advance Preparations .......................................................................... 14 3.2.3 Time Planning ....................................................................................... 14 3.2.4 Utilization of Reagents ......................................................................... 16 3.3 First Strand Synthesis .................................................................................... 16 3.4 Second Strand Synthesis ............................................................................... 18 3.5 Sal I Adapter Addition ..................................................................................... 18 3.6 Not I Digestion ................................................................................................ 19 3.7 Column Chromatography ............................................................................... 20 3.8 Ligation of cDNA to the Vector ....................................................................... 21 3.9 Introduction of Ligated cDNA into E. coli by Transformation ..................................................................... 24 3.10 Introduction of Ligated cDNA into E. coli by Electroporation ...................................................................... 24 3.11 Analysis of cDNA Products ............................................................................ 25 3.11.1 First Strand Yield ................................................................................. 25

3.11.2 Second Strand Yield ............................................................................ 26 3.11.3 Gel Analysis ......................................................................................... 26 3.11.4 Analysis of cDNA from the cDNA Size Fractionation Column ........... 27

4. Troubleshooting .......................................................................................29 4.1 Isolation of mRNA ............................................................................................29 4.2 First Strand Reaction .......................................................................................29 4.3 Second Strand Reaction .................................................................................29 4.4 Sal I Adapter Addition and Not I Digestion ......................................................30 4.5 Column Chromatography ................................................................................30 4.6 Ligation of cDNA to the Vector ........................................................................30 4.7 Introduction of Ligated cDNA into E. coli ........................................................31

5. Additional Protocols ...............................................................................32 5.1 Expansion of Plasmid cDNA Libraries ............................................................32

6. References ..................................................................................................34

7. Related Products ......................................................................................35

Figures1. Summary of the SuperScript® Plasmid System Procedure .......................................42. Sequence of the Not I Primer-Adapter .......................................................................73. Sequence of the Sal I Adapter ....................................................................................74. The BP Reaction .........................................................................................................95. MapandMultipleCloningSiteofPlasmidpCMV•SPORT6 ....................................116. MapandMultipleCloningSiteofPlasmidpSPORT1 .............................................127. Detailed Protocol Flow Diagram ...............................................................................158. Alkaline Agarose Gel Analysis of First and Second Strand cDNA Synthesized with the SuperScript® Plasmid System.....................................279. Electrophoretic Analysis of Size-Fractionated cDNA ...............................................28

Table1. CommonFeaturesofpSPORT1andpCMV•SPORT6 ............................................9

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Table of Contents

1Notices to Customer

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1.1 Important InformationThis product is authorized for laboratory research use only. The product has not been qualified or found safe and effective for any human or animal diagnostic or therapeutic application. Uses for other than the labeled intended use may be a violation of applicable law.

1.2 Precautions for UseWarning: This product contains hazardous reagents. It is the end-user’s responsibility to consult the applicable SDS(s) before using this product. Disposal of waste organics, acids, bases, and radioactive materials must comply with all appropriate federal, state, and local regulations. If you have any questions concerning the hazards associated with this product, please call the Life Technologies, Inc. Environmental Health and Safety Chemical Emergency hotline at (301) 431-8585.

1.3 Limited Label LicensesLimited Label License No. 358: Research Use OnlyThe purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser’s activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@lifetech.com orOut Licensing, LifeTechnologies, 5791VanAllenWay, Carlsbad, California 92008.

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1.4 TrademarksThe trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.Gene Pulser® is a registered trademark of Bio-Rad Laboratories, Inc.

RNase AWAY™ is a trademark of Molecular BioProducts, Inc.

SeaPrep® is a registered trademark of Lonza Sales AG Company.

1.5 DisclaimerLIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.

©2013 Life Technologies Corporation. All rights reserved.

Notices to Customer

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Overview

2.1 cDNA LibrariesA cDNA library is an array of DNA copies of an mRNA population that are propagated in a cloning vector and usually maintained in E. coli. Every good cDNA library has three key characteristics that distinguish it from mediocre counterparts:1. It is large enough to contain representatives of all sequences of interest, some

of which may be derived from low-abundance mRNAs.2. It includes a minimal number of clones that contain small (often defined

arbitrarily as ≤500 bp) cDNA inserts.3. It is composed of cDNA inserts that are near full-length copies of the mRNAs

from which they were derived.

Because a cDNA library is the end product of many individual steps, the quality can be compromised by inefficiency at any point in the procedure. The SuperScript® Plasmid System for cDNA Synthesis and Plasmid Cloning with Gateway® Technology integrates state-of-the-art cDNA synthesis with simplified downstream procedures to produce cDNA ligated to one of several versatile plasmid vectors provided, ready to introduce into E. coli. Use of a cloning vector containing attB sites creates a Gateway®-compatible library that allows entry into the Gateway® system for fast cloning and expression of DNA segments using recombinational cloning (see Section 2.6). If the starting material is of high quality, the cDNA library constructed with this system will satisfy all three of the preceding criteria.

cDNA libraries can be broadly classified as directional or random. All members of a directional library contain cDNA inserts cloned in a specific orientation relative to the transcriptional polarity of the original mRNAs; members of random libraries contain cDNA inserts cloned in either orientation. Directional libraries are usually constructed to drive expression of the cloned gene by a controllable promoter (contributed by the vector) and to immunologically detect the target protein (an antigen) with a specific probe (an antibody). Unlike random libraries, in which only ~50% of the clones contain cDNA inserts oriented properly for expression, all members of a directional library are potentially able to express antigen. Thus, cDNAs can be detected by screening hal f as many clones. This 2-fold reduction in time and resources needed for detection is particularly beneficial in the case of rare cDNAs, which may require screening of as many as 106 clones.

Directional cDNA cloning facilitates the construction of subtracted libraries. A subtracted library is the end product of a comparison between two mRNA populations, which is enriched for sequences that are present in one population, but not the other (1). The two populations may come from different cell or tissue types; they also may come from the same cell or tissue at various stages of differentiation or under altered physiological conditions that affect the transcriptional pattern within the cell. Although there are different methods to produce subtracted libraries, they all use hybridization to remove sequence information common to the two populations in question.

The major steps in constructing a directional cDNA library from an mRNA population using the SuperScript® Plasmid System are summarized in figure 1.

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2.2 mRNA IsolationConstruction of a good cDNA library begins with the preparation of high quality mRNA. The quality of the mRNA dictates the maximum amount of sequence information that can be converted into cDNA. Thus, it is important to optimize the isolation of mRNA from a given biological source and to prevent adventitious introduction of RNases into a preparation that has been carefully rendered RNase-free. For optimal results, the mRNA must be purified over an affinity column [oligo(dT) cellulose being the most commonly used matrix] to select the polyadenylated [poly(A)+] RNA (2). Since the vast majority of mRNA is poly(A)+, this selection operationally defines the mRNA population. Typically, 0.5%—2% of a total RNA population is mRNA, so isolation of this fraction from rRNA, tRNA, and degraded mRNA enhances the synthesis of first strand cDNA and minimizes spurious transcription of non-mRNAs. An mRNA preparation that has undergone two selections over this matrix (in which the eluate from one round of purification has been bound to the column and eluted a second time) will produce the highest quality mRNA. When properly prepared, oligo(dT) cellulose-purified RNA will be ≥90% mRNA.

Figure 1. Summary of the SuperScript® Plasmid System procedure.

OverviewAAAAAATTTTTTNot I primer-adapter

AAAAAATTTTTT

First strand synthesis

AAAAAATTTTTT

Second strand synthesis

AAAAAATTTTTT

Not I

AAAAAATTTTTT

Size fractionation

AAAAAATTTTTT

Ligation to Not I-Sal I-cut vector

Sal I adapter addition

Not I digestion

Not I

Not I

Not I

Sal I Sal I

Sal I Not I

mRNA

cDNA ready for transformation

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The amount of mRNA needed to prepare a library is dependent on the efficiency of the individual steps needed to convert the mRNA into a form that can be cloned and on the efficiency with which the recombinant molecules can be introduced into a host. Generally, 1 to 5 µg of mRNA will be sufficient to construct a cDNA library containing 105 to 106 clones in E. coli.

2.3 First Strand SynthesisSuperScript® II RT is a modified version of Moloney Murine Leukemia Virus (M-MLV) RT with reduced RNase activity (3,4). This modification is significant because RNase H activity is detrimental to the first strand cDNA synthesis reaction in two ways:1. The initiation of first strand synthesis depends upon the hybridization of a

primer to the mRNA, usually at the poly(A) tail. This hybrid is a substrate, not only for the polymerase activity of the RT, but also for the RNase H activity (5). In the resulting competition between these two activities, the extent to which the RNase H activity destroys the hybrid prior to the initiation of polymerization determines the maximal number of initiation events that can actually occur. Hydrolysis of the RNA in the hybrid reduces the maximal yield of cDNA by effectively removing a portion of the mRNA from the reaction.

2. When the RT is synthesizing the first strand cDNA, the RNase H activity will quickly degrade the template that has already been copied because the mRNA is in hybrid form as a result of the polymerization reaction. If the scissions in the mRNA occur near the point of chain growth, the uncopied portion of the mRNA can dissociate from the transcriptional complex, resulting in termination of cDNA synthesis for that template and consequent reduction in the yield of full-length cDNA. This problem can be exacerbated if the RT pauses during transcription at certain primary or secondary structural domains.

When used with synthetic RNA produced in vitro, SuperScript® II RT has demonstrated significantly greater full-length cDNA synthesis and higher yields of first strand cDNA than M-MLV RT (6,7).

The reaction conditions for first strand synthesis catalyzed by SuperScript® II RT have been optimized for yield and size of the cDNAs. The optimal first strand reaction temperature for SuperScript® II RT is 37°C; however, should secondary structure make reverse transcription difficult, a higher reaction temperature may be used. SuperScript® II RT is stable at 45°C to 50°C and can be used at this temperature, if necessary.

The amount of mRNA can be as high as 5 µg in a 20-µL first strand cDNA synthesis reaction. We recommend using at least 1 µg of mRNA so that there will be sufficient material at the end of the procedure to obtain the required number of clones. The amount of SuperScript® II RT needed in the first strand reaction varies linearly with the amount of mRNA: 200 units of SuperScript® II RT for ≤1 µg of mRNA, and 200 units per µg for 1 to 5 µg of mRNA. Although the exact ratio of SuperScript® II RT to mRNA is not critical, these approximate proportions have produced reliable results.

2.4 Second Strand SynthesisThe primary sequence of the mRNA is recreated as second strand cDNA using the first strand cDNA as a template. The SuperScript® Plasmid System uses nick translational replacement of the mRNA to synthesize the second strand cDNA. First describedbyOkayamaandBerg(8),andlaterpopularizedbyGublerandHoffman(9), second strand synthesis is catalyzed by E. coli DNA polymerase I in

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Overview combination with E. coli RNase H and E. coli DNA ligase. Although RNase H is not essential if the first strand synthesis is catalyzed by AMV or M-MLV RT, E. coli RNase H must be included in the second strand reaction when SuperScript® II RT has been used for first strand cDNA synthesis. E. coli DNA ligase has been shown to improve the cloning of double-stranded (ds) cDNA synthesized from longer (≥2 kb) mRNAs (10).

The first and second strand syntheses are performed in the same tube without intermediate organic extraction or ethanol precipitation. This one-tube format speeds the synthesis procedure and maximizes recovery of cDNA. The efficiency of the second strand reaction is influenced by the amount and concentration of the reactants, so the instructions must be followed as described for best results. The second strand reaction is incubated at 16°C to prevent spurious synthesis by DNA polymerase I due to its tendency to strand-displace (rather than nick translate) at higher temperatures. The last step in the cDNA synthesis procedure is to ensure that the termini of the cDNA are blunt. This is easily done by adding T4 DNA polymerase to the second strand reaction mixture and incubating briefly at 16°C. The cDNA is then deproteinized by organic extraction and precipitated with ethanol to render it ready for downstream manipulation.

2.5 Constructing Directional Libraries2.5.1 Introducing Asymmetry into cDNA

Directionality is obtained by introducing asymmetry at the ends of cDNA, typically by introducing two different restriction endonuclease sites at the termini. Several strategies can achieve this end, but none is simpler than using a primer-adapter to initiate first strand synthesis. A primer-adapter is a primer for reverse transcription that also encodes sequence information for one or more restriction sites; the resulting cDNA retains the restriction endonuclease recognition sites. The restriction sites introduced into cDNA by the primer-adapter provide the asymmetric element needed to obtain directional clones. After cDNA synthesis, the restriction-site terminus is exposed by digestion with the appropriate restriction endonuclease. The resulting terminus identifies the end of the cDNA corresponding to the 3´ end of the mRNA.

The SuperScript® Plasmid System features a Not I primer-adapter designed to prime first strand cDNA synthesis at the poly(A) tail of mRNAs. This primer-adapter, 44 bases in length, contains 15 dT residues and 4 restriction endonuclease sites chosen for their relative rarity in mammalian genomes. To complement the Not I primer-adapter, the system also includes the restriction endonuclease Not I. The 8-base recognition sequence of this enzyme is extremely rare in most cDNAs, occurring approximately once in 106 bp. The sequence and organization of the Not I primer-adapter are shown in Figure 2.

2.5.2 Maximizing the Ligation Efficiency of cDNA to the Vector by Adapter Addition

The product of the first and second strand reactions produced with the SuperScript® Plasmid System is blunt-ended cDNA. Although restriction digestion exposes the 5´ extension of the Not I site, the other end of the cDNA remains blunt-ended and is thus a poor substrate for T4 DNA ligase. To maximize ligation efficiency into the vector, the blunt end of the cDNA is converted to a terminus that contains a 5´ extension by adding adapters to the cDNA. Adapters are short, duplex oligomers that are blunt-ended at one terminus and contain a 4-base, 5´ extension at the other terminus. The blunt-end ligation of the adapters to the cDNA can be driven by adapter excess, much the way molecular linkers are added to DNA. However, unlike linkers, adapters contain preformed extensions and do not require restriction

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digestion to expose the termini.

The 4-base, 5´ extension of the adapters provided with the SuperScript® Plasmid System corresponds to the termini produced by several restriction endonucleases, including Sal I and Xho I. Because ligation to a Sal I terminus will recreate a Sal I recognition site, we refer to these adapters specifically as Sal I adapters. The sequence of the Sal I adapter included in the SuperScript® Plasmid System is shown in figure 3. Several details should be noted:1. The recognition sequence for MLu I is contained within the Sal I adapter.

Because the CpG motif is repeated twice within the MLu I recognition sequence, it is a relatively rare site in mammalian genomes, despite being only 6 bases in length. The cloning vector used in the SuperScript® Plasmid System (see Section 2.6, The Cloning Vector: Plasmid pSPORT 1, for details) contains an additional MLu I site positioned so that plasmids purified from clones can be digested with MLu I to release the cDNA insert.

2. Only one of the oligomers of theSal I adapter is phosphorylated, which eliminates self-ligation of the adapters during ligation to the cDNA.

In practice, the Sal I adapters are added to the cDNA prior to digestion with Not I, which places the same Sal I 5´-extension at both ends of the cDNA. At this point, the cDNA could be used to construct a random library by phosphorylating the cDNA and ligating it to a Sal I-digested vector that has been dephosphorylated to reduce the background arising from self-ligation of the vector. If you are interested in constructing random libraries, consult Molecular Cloning: A Laboratory Manual (11) for details on preparing phosphorylated DNA and dephosphorylated vectors.

2.5.3 Size Fractionation of cDNASize fractionation of cDNA, following adapter addition and digestion with Not I, is important for several reasons. Residual adapters are present in large molar excess and can impede vector ligation to cDNA by ligating to the Sal I termini of the

predigested vector. Additionally, the fragments released from the cDNA by Not I digestion have Sal I termini at one end and Not I termini at the other, and can contaminate the library with apparently “empty” clones, which increases the screening burden.

Size fractionation also reduces the tendency of smaller (<500 bp) inserts to predominate the library. These smaller cDNAs can arise for several reasons:1. Most mRNA preparations are not size-selected, so partially degraded mRNAs

can be selected on the oligo(dT) cellulose columns along with longer mRNAs. These will be reverse transcribed into small cDNAs.

2. If extreme care is not taken to prevent RNase contamination during first strand synthesis, degradation can occur when the mRNA is manipulated.

Figure 3. Sequence of the Sal I adapter.

5´-TCGACCCACGCGTCCG-3´3´- GGGTGCGCAGGCp-5´

Sal I MLu I

Figure 2. Sequence of the Not I primer-adapter.

5´-pGACTAGT TCTAGA TCGCGA GCGGCCGC CC (T)15-3´ Spe I Xba I Nru I Not I

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Overview 3. Some mRNAs contain regions that are not readily reverse transcribed, and RT is not able to synthesize complete first strands.

Column chromatography is the simplest method of producing size-fractionated cDNA, free of adapters and other low molecular weight DNAs. The SuperScript® Plasmid System contains three 1-mL, prepacked, disposable columns that quickly and easily remove cDNAs <500 bp and size-fractionate cDNAs >500 bp, thus facilitating construction of libraries from fractions enriched for larger cDNA. The column chromatography buffer (described in Section 3) is formulated to allow the cDNA in the column fractions to be ligated directly into the predigested plasmid cloning vector (Not I-Sal I-Cut), which minimizes the risk of losing precious cDNA in an ethanol precipitation step. Although the final yield of size-fractionated cDNA will depend upon the recovery at each step of the procedure, the average overall yield should be 5%—10% of the mass of the starting mRNA.

2.5.4 Ligation of Size-Fractionated cDNA to the Plasmid Vector and Introduction into E. coli

The ligation reaction described in Section 3 will suffice for most applications. We have found that 10—20 ng of cDNA saturates the 50 ng of vector in the ligation reaction and that to use more cDNA is wasteful (as little as 1 ng of cDNA can be used in the ligation reaction). However, it is possible that for a particular population of cDNA, this ratio may not be optimal. If the described ligation conditions do not yield enough transformants to make your library complete, we recommend optimization of the reaction by varying the ratio of vector to cDNA and determining which ratio yields the maximal number of clones.Ofcourse, the number of transformants is dependent upon the transformation efficiency of the E. coli cells used to plate the library. If the cells yield ~1 × 109 transformants/µg of pUC19 plasmid DNA, then the ligated cDNA should yield 0.5 to 1 × 107 transformants/µg of vector; this is equivalent to 2.5 to 5.0 × 107

transformants/µg of cDNA. Thus, a library containing 5 × 105 clones can be constructed from as little as 20 ng of cDNA.

The ligated cDNA can also be introduced into E. coli cells by electroporation, which generally will yield a greater number of transformants from the same amount of ligated cDNA (2.5 × 108 to 1 × 109 transformants/µg of cDNA). Electroporation may be especially useful if the library must be very large or if you have <10 ng of cDNA.

2.6 Gateway® Cloning TechnologyGateway® Cloning Technology provides a powerful, flexible approach for transferring DNA segments between vectors using site-specific recombination. The cDNA library vectors pCMV•SPORT6 and pEXP-AD502 (ProQuest™Two-Hybrid) have beenconstructed to contain the Gateway® site-specific recombination sites (attB1 and attB2) flanking the multiple cloning site into which the cDNA is cloned. Therefore, clones isolated from these libraries can be rapidly transferred into other Gateway® vectors using site-specific recombination rather than restriction endonucleases and ligase (Figure 4). The speed and ease of Gateway® cloning facilitate DNA sequencing, generating RNA probes, or expressing proteins from the cDNA clone. Details describing the mechanism, applications, and protocols (including a Gateway® manual) can be obtained at www.lifetechnologies.com.

2.7 Cloning VectorsThe cloning vectors included in the SuperScript® Plasmid System are plasmids pCMV•SPORT6 and pSPORT1. They both have been engineered to includeseveral useful performance features (Table 1).

Both plasmids are supplied predigested with Sal I and Not I and have been carefully prepared to provide a low background of nonrecombinant colonies.

Note: If the cDNA library needs to be expanded, use the semi-solid culture method provided in the Additional Protocols section. This procedure will minimize representational biases that could occur during growth in liquid broth.

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Table 1. Common features of pSPORT 1 and pCMV•SPORT6.

Cloning site: Sal I and Not I cloning sites for directional cDNA cloning

Replicon: colE1Antibiotic resistance: Ampicillinf1 intergenic region for production of ss DNABacteriophage SP6 and T7 RNA polymerase promoters: for in vitro RNA synthesis from either strand

In vitro transcription from phage promoters. The SP6 and T7 RNA polymerase promoters can be used to synthesize RNA in vitro from purified plasmid DNA using either SP6 or T7 RNA polymerase. These promoters oppose each other and bracket the cloning site. Thus, either strand of the cDNA clone can be copied into RNA. The RNA can be used as probes, for in vitro translational studies, or in the construction of subtracted libraries.

Production of single-stranded DNA. The bacteriophage intergenic (IG) region on the plasmids allows synthesis of the single-stranded form of the plasmid. In this procedure, plasmids containing cDNA inserts are introduced into a strain of E. coli containing the F´ episome, and the cells are then infected with a helper phage such asM13KO7 (13).The plasmids are rescued as single-stranded circles that aresecreted into the growth medium as phage-like particles. Following purification and DNA extraction by established techniques, the DNA from the particles can be used for dideoxy sequencing or site-directed mutagenesis (14). The strand that is produced from this plasmid is the equivalent of the (+) strand of a single-stranded phage such as M13 (and the strand corresponding to the mRNA from the cloned cDNA). Since both plasmids contain the f1 intergenic region, they can be used for the preparation of substrates for screening.

Keydifferences betweenpSPORT1andpCMV•SPORT6 are the promoters forexpression of cDNA and the additional benefits of Gateway® Technology available in pCMV•SPORT6.

2.7.1 PlasmidpCMV•SPORT6

Plasmid pCMV•SPORT6 was constructed for theGateway® Cloning System. Unique performance features include:

Gateway® compatibility. This vector contains the Gateway® attB1 and attB2 recombination sites flanking the multiple cloning site. The cDNA present in clones isolated from pCMV•SPORT6 libraries can be transferred into otherGateway®-compatible vectors for sequencing, protein expression, and functional analysis.

Figure 4. The BP reaction.

ExpressionClone

By-product

Donor Vector

attB1 attB2attP1 attP2

attL1 attL2

attR1 attR2

Apr Kmr

AprKmr

ccdBGene

Gene ccdB

X

+

BP CLONASE™ Enzyme Mix

EntryClone

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Overview Note: Because the 5´-ends of cDNAs are random, only those clones in the proper reading frame will be directly useful for protein expression.

Expression of cloned cDNA. The CMV promoter enables the transient expression of cloned cDNAs in eukaryotic cells. Libraries and individual clones may be screened by functional analysis using this eukaryotic promoter. Unlike the pSPORT1 vector, the pCMV•SPORT6 vector does not contain the lac promoter (lacP) and so can not be used to express cloned genes in E. coli. See figure 5 for a diagram of the multiple cloning site.

2.7.2 Plasmid pSPORT 1

Unique performance features include :

Expression of cloned cDNA. PlasmidpSPORT1canbeusedtoexpressclonedgenes by inducing the lac promoter (lacP). The vector contains a single copy of the lac repressor gene (lacI); transcription of cDNA inserts from the promoter is effectively repressed unless the promoter is induced by the addition of 1 mM isopropylthio-ß-galactoside (IPTG) to plates or liquid media. Libraries are not plated under inducing conditions unless expression is specifically desired. Inducing expression can lead to loss of clones from the library due to accumulation of proteins that may interfere with the metabolism of E. coli.

Blue-white screening of colonies. ContainsthelacOPZ´sequenceforblue-whitescreening. This allows simplified selection of positive inserts.

Construction of nested deletions. The multiple cloning site (MCS) has been designed to facilitate the construction and sequencing of nested deletions using the method of Henikoff (12). The restriction sites that yield 3´ and 5´ extensions are clustered to provide flexibility for the unidirectional digestion of the DNA. See figure 6 for a diagram of the multiple cloning site.

2.7.3 Plasmid pSPORT 2PlasmidpSPORT2isavailableseparatelyandisfunctionallysimilartopSPORT1,except that the Not I - Sal I portion of the MCS is inverted, thus reversing the cDNA orientationrelativetothecDNAcloneddirectionallyintopSPORT1.pSPORT1andpSPORT2makeasuitablevectorcombinationforsubtractivehybridization.

2.7.4 Plasmid pEXP-AD502Plasmid pEXP-AD502 is a Gateway®-compatible vector for construction of cDNA libraries as ProQuest™Two-HybridActivation Domain Fusions. This vectorcontains attB1 and attB2 sites flanking the multiple cloning site, with the attB1 site in frame with the Activation Domain. Clones (AD-Y) isolated following a two-hybrid screen using a pEXP-AD502 cDNA library can be rapidly transferred using Gateway® technology into other vectors for protein expression and functional analysis, thereby facilitating validation of candidate interactors. Additional informationontheProQuest™ Two-Hybrid System with Gateway® Technology can be obtained from Technical Services or at www.lifetechnologies.com.

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Figure5.MapandMultipleCloningSiteofPlasmidpCMV•SPORT6.

2Cla I 122 Hpa I 257

Mun I 268Mam I 356

Bcl I 361

Nco I 1227SnaB I 1249

Nsi I 1534

Sap I 1807BspLU11 I 1930

AlwN I 2341

Eam1105 I 2860

Bsa I 2932

Sca I 3343Bcg I 3381

Xmn I 3460

PflM I 3846

Dra III 4159NgoA IV 4265

pCMV•SPORT 64396 bp

SV40polyadenylation signal

T7 promoter

SP6 promoter

CMV promoter

ori

lox P

Apr

incA

f1 intergenicregion

Nhe I 637

Stu I 872Sst II 918Avr II 924Bbs I 960Esp3 I 992

att B1 att B2

multiplecloningsiteKpn I 822

Mlu I 736

pCMV•SPORT 6 multiple cloning site and primer binding regions: 641-917 (The sequence listed here is the (-) strand.)

5’-GCAGTTTTCC CAGTCACGAC GTTGTAAAAC GACGGCCAGT GCCTAGCTTA TAATACGACT CACTATAGGG ACCACTTTGT ACAAGAAAGC TGGGTACGCG TAAGCTTGGG CCCCTCGAGG GATACTCTAG AGCGGCCGCCC

Sal I EcoR V Sma I EcoR I Rsr II Kpn I

Mlu I Hind III Apa I Xho I Xba I Not I

CGGACGCG TGGGTCGACG ATATCCCGGG AATTCCGGAC CGGTACCAGC CTGCTTTTTT GTACAAACTT GTTCTATAGT GTCACCTAAA TAGGCCTAAT GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCT-3’

Mlu I* PinA I

700•

800•

M13/pUC Forward 23-Base Sequencing Primer

M13/pUC Reverse 23-Base Sequencing Primer

T7 Promoter Primer

cDNA Insert

5’-CC CAGTCACGAC GTTGTAAAAC G-3’→

←3’-GATATCA CAGTGGATTT A-5’SP6 Promoter Primer

* This Mlu I restriction site is contained within the Sal I adapter introduced into the vector upon ligation of the cDNA insert.

←3’-GGACAC ACTTTAACAA TAGGCGA-5’

5’-TAATACGACT CACTATAGGG-3’→

I---------- T7 promoter ------------I I----------------- att B2 ---------------------------I

I-------- SP6 promoter -----------I

I--------------- att B1 --------------I

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Overview

Figure 6. Map and Multiple Cloning Site of Plasmid pSPORT 1.

Xmn I 3329

Aat II 191

Esp3 I 582

Bsa I 2801

Nar I 552

Eam1105 I 2729

Sca I 3212

AlwN I 2252

pSPORT 14109 bp

Apr

lacOPZ

lacI

Apa I 984

Hpa I 687

Dra III 3872

EcoR V 743

Pfl M I 1610

ori

Bcl I 1177

Pst I 280

NgoA IV 3978

SP6 promoter

T7 promoter

Sap I 1718

BstE II 1009

lacI promoter

multiplecloningsite

f1 intergenicregion

pSPORT 1 multiple cloning site and primer binding regions: 125-372

5-CCCAGT CACGACGTTG TAAAACGACG GCCAGTGAAT TGAATTTAGG TGACACTATA GAAGAGCTAT GACGTCGCAT GCACGCGTAC GTAAGCTTGG ATCCTCTAGA GCGGCCGCCC CGGACGCGT Aat II* Sph I Mlu I SnaB I BamH I Not I

GGGTCGACCC GGGAATTCCG GACCGGTACC TGCAGGCGTA CCAGCTTTCC CTATAGTGAG TCGTATTAGA GCTTGGCGTA ATCATGGTCA TAGCTGTTTC CTGTGTGAAA TTGTTATCCG CT-3

Mlu I**

150•

300•

M13/pUC Forward 23-Base Sequencing Primer

M13/pUC Reverse 23-Base Sequencing Primer

SP6 Promoter Primer5-CCCAGT CACGACGTTG TAAAACG-3→

←3-GG GATATCACTC AGCATAAT-5

T7 transcription start

←3-G GACACACTTT AACAATAGGC GA-5

5-ATTTAGG TGACACTATA G-3→

I-------- SP6 promoter ------I

I--------- T7 promoter ---------I

cDNA Insert

* The Mlu I restriction site is contained within the Sal I adapter introduced into the vector upon ligation of the cDNA insert.*Requires increased enzyme:DNA for complete digestion.

350•

200•�SP6 transcription start

Hind III Xba I

Xma III

Sal IAcc I

� T7 Promoter Primer

α-peptide start

EcoR ISma I Rsr II Kpn I Pst I

Kpn2 I Sse8387 IPinA I

Sun I

I�

3

13

3.1 ComponentsThe components of the SuperScript® Plasmid System with Gateway® Technology for cDNA Synthesis and Cloning are as follows. Components are provided in sufficient quantities to perform three separate experiments, each converting up to 5 µg of mRNA into size-fractionated, plasmid-ligated cDNA, ready for transformation or transfection into E. coli. Sore chromatography columns (Part No. 8092CL) at 2°C to 8°C and store the reagent assembly (Part No. 8248RT) at –30°C to –10°C.

Component AmountNot I primer-adapter (0.5 µg/µL) .......................................................................................... 8 µL5X first strand buffer [250 mM Tris-HCl (pH 8.3),

375 mM KCl, 15 mM MgCl2] ........................................................................................ 1 mL0.1 M DTT ......................................................................................................................... 250 µL10 mM dNTP mix (10 mM each dATP, dCTP, dGTP, dTTP) ............................................... 20 µLSuperScript® II RT (200 units/µL) ....................................................................................... 50 µL5X second strand buffer [100 mM Tris-HCl (pH 6.9), [450 mM KCl,

23 mM MgCl2, 0.75 mM ß-NAD+, 50 mM (NH4)2SO4] .............................................. 500 µLE. coli DNA ligase (10 units/µL) .......................................................................................... 10 µLE. coli DNA polymerase I (10 units/µL) .............................................................................. 50 µLE. coli RNase H (2 units/µL) ............................................................................................... 20 µLT4 DNA polymerase (5 units/µL) ........................................................................................ 10 µL5X T4 DNA ligase buffer [250 mM Tris-HCl (pH 7.6),

50 mM MgCl2, 5 mM ATP, 5 mM DTT, 25% (w/v) PEG 8000] ..................................... 1 mLSal I Adapters (1 µg/µL) ..................................................................................................... 50 µLT4 DNA ligase (1 unit/µL) ................................................................................................... 50 µLREact® 3 Buffer ................................................................................................................... 1 mLNot I (15 units/µL) ............................................................................................................... 12 µLDEPC-treated water ....................................................................................................... 1.25 mLplasmidpCMV•SPORT6,Not I-Sal I-Cut (50 ng/µL) .......................................................... 50 µLplasmidpSPORT1,Not I-Sal I-Cut (50 ng/µL) .................................................................. 50 µLcontrol RNA (0.5 µg/µL) ..................................................................................................... 15 µLyeast tRNA (1 µg/µL) ........................................................................................................ 100 µLcDNA size fractionation columns ......................................................................................... threemanual ................................................................................................................................... one

3.2 General Comments3.2.1 mRNA PurificationOne of the most important steps preceding the synthesis of cDNA and theestablishment of a library is isolation of intact mRNA. The FastTrack® 2.0 mRNA Isolation Kit allows isolation of intact mRNA essentially free of ribosomal RNA in a fast and convenient procedure.

Successful cDNA synthesis demands an RNase-free environment at all times, which will generally require the same level of care used to maintain aseptic conditions when working with microorganisms. Several additional guidelines should be

Methods

14

followed:

1. Never assume that anything is RNase-free, except sterile pipets, centrifuge tubes, culture tubes, and any similar labware that is explicitly stated to be sterile.

2. Dedicate a separate set of automatic pipets for manipulating RNA and the buffers and enzymes used to synthesize cDNA.

3. Avoid using any recycled glassware unless it has been specifically rendered RNase-free by rinsingwith 0.5 NNaOH followed by copious amounts ofautoclaved, distilled water. Alternatively, bake glassware at 150°C for 4 hours.

4. Microcentrifuge tubes can generally be taken from an unopened box, autoclaved, and used for all cDNA work. If necessary, soak the tubes overnight in a 0.01% (v/v) aqueous solution of diethylpyrocarbonate (DEPC); rinse them with autoclaved, distilled water; and autoclave them.

5. If made with RNase-free labware, most solutions can be made from reagent-grade materials and distilled water and then autoclaved. Prepare heat-sensitive solutions using autoclaved, distilled water, and filter them to 0.2 µm using sterile, disposable filterware.

6. If all else fails, most aqueous buffer solutions can be treated with 0.01% (v/v) DEPC and autoclaved.

3.2.2 Advance PreparationsBefore using this system, please review the protocol flow diagram in figure 7. You will need the following items not included in this system:• autoclaved 1.5-mL microcentrifuge tubes• microcentrifuge capable of generating a relative centrifugal force of 14,000 × g• automatic pipets capable of dispensing 1–20 µL, 20–200 µL, and 200 µL to 1 mL• autoclaved, disposable tips for automatic pipets• disposable gloves• 16°C and 37°C water baths• 1–10 µCi [α-32P]dCTP (400–3000 Ci/mmol)• 500 mL 10% (w/v) TCA (trichloroacetic acid) containing 1% (w/v) sodium

pyrophosphate (store at 4°C)• glass fiber filters (1 × 2 cm) (Whatman GF/C or equivalent)• buffer-saturated phenol:chloroform:isoamyl alcohol [25:24:1 (v/v/v)]• TEN buffer [10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 25 mM NaCl; autoclaved]• 7.5 M ammonium acetate (NH4OAc)filteredthroughasterile,0.2-µmnitro-

cellulose filter• 70% (v/v) ethanol (–20°C)

3.2.3 Time PlanningStarting with poly(A)+ RNA, these protocols are designed to yield vector-ligated cDNA, ready for transformation or electroporation, in ~3 days. For best results, the procedure should be completed as quickly as possible because radiochemical effects induced by the decay of the 32P in the cDNA can diminish transformation efficiencies over time.

We recommend that the protocols be completed as follows:

Day 1: Sections 3.3 and 3.4, and steps 1 and 2 of Section 3.5 (overnight incubation of Sal I Adapter Addition reaction).

Day 2: Complete Sections 3.5, 3.6, and 3.7 (store overnight at –20°C).Day 3: Sections 3.8 and 3.9 or 3.10.

If it is necessary to interrupt the procedure at any other point, you may do so following any of the organic extractions described in the individual protocols. When

Note: The use of Gibco BRL Phenol: Chloroform:Isoamyl Alcohol [25:24:1 (v/v/v)] is recommended. If making your own, saturate the redistilled phenol with TEN buffer, not with distilled water.

Note: Suggested stopping points are noted in the protocol with the word: Stop.

Note: Buffers containing primary amines (such as Tris) cannot be effectively treated by this method.

Overview

3

15

Figure 7. Detailed protocol flow diagram.

Poly(A)+ mRNA

Not I primer-adapterDEPC-treated water

5X first strand buffer0.1 M DTT10 mM dNTP mix[α-32P]dCTP

SuperScript® II RT

First strand reaction

DEPC-treated water5X second strand buffer10 mM dNTP mixE. coli DNA ligaseE. coli DNA polymerase IE. coli RNase H

5X T4 DNA ligase bufferNot I-Sal I-Cut vectorDEPC-treated waterT4 DNA ligase

Transfer to ice

First strand cDNA

cDNA library cDNA library

T4 DNA polymerase

Extract, precipitate

Second strand reaction

70°C, 10 min

37°C, 2 min

37°C, 1 h

16°C, 2 h

16°C, 5 min

DEPC-treated water5X T4 DNA ligase bufferSal I adaptersT4 DNA ligase

Extract, precipitate

ds cDNA

DEPC-treated waterREact 3 bufferNot I

Extract, precipitate

Column chromatography

Sal I-adapted cDNA

Vector-ligated cDNA

37°C, 2 h

Room temperature, 3 h or overnight, 4°C

Transform E. coli Precipitate, electroporateE. coli

16°C, 16 h

Remove aliquot for yield and gel electrophoretic analyses

Size-fractionated cDNAwith Not I-Sal I termini

16

stopping at any such point, always store the cDNA at –20°C as the uncentrifuged ethanol precipitate, to minimize the aforementioned effects of 32P decay.

3.2.4 Utilization of ReagentsReagents included with the SuperScript® Plasmid System are provided in sufficient quantities to perform three complete experiments converting up to 5 µg of mRNA into size-fractionated, plasmid-ligated cDNA. Additionally, the components for first and second strand synthesis are provided in sufficient quantities to perform Sections 3.3 and 3.4 four times. You may wish to use these extra quantities to test a small amount of your mRNA by determining first or second strand yield and visualizing the distribution of the products by gel electrophoresis. Alternatively, the extra components can be held as a backup in case of accidental loss of material or procedural error.

3.3 First Strand SynthesisThe 20-µL reaction described is designed to convert up to 5 µg of mRNA into first strand cDNA. The amount of SuperScript® II RT added to the reaction will be dependent upon the amount of starting mRNA. We recommend 200 units of SuperScript® II RT for ≤1 µg of mRNA, and 200 units/µg of mRNA for 1–5 µg of mRNA. Note: For pEXP-AD502 cDNA libraries, use 5 µg of mRNA as starting material to obtain the required 30 ng cDNA.

If second strand cDNA is to be labeled instead of first strand cDNA, the first strand reaction should be set up without [α-32P]dCTP (adjust the amount of water in the reaction to maintain the 20-µL final volume), and the reaction should be carried through the second strand synthesis procedure as described in Section 3.4. In this case, add 1 µL (10 µCi/µL) [α-32P]dCTP to the second strand reaction after the 10 mM dNTP mix is added.

A control RNA is included in the SuperScript® Plasmid System as an aid in verifying that the system performs in your hands. If you decide to use the control RNA, simply substitute 4 µL (2 µg) in the first strand reaction for your mRNA.1. Add 2 µL of Not I primer-adapter to a sterile 1.5-mL microcentrifuge tube. Add

mRNA, diluted as needed with DEPC-treated water, according to the following table:

µg of mRNA

≤1 2 3 4 5

mRNA (plus DEPC-treated water) (µL) 9 8 7 6 5 Total volume (µL) 11 10 9 8 7

2. Heat the mixture to 70°C for 10 minutes, and quick-chill on ice. Collect the contents of the tube, by brief centrifugation, and add the following:

Component ......................................................... Volume (µL)5X First Strand Buffer .............................................................40.1 M DTT ...............................................................................210 mM dNTP Mix ....................................................................1[α-32P]dCTP (1 µCi/µL) ...........................................................1

The total volume should now correspond to the following table:

µg of mRNA (from step 1)

≤1 2 3 4 5

Total volume (µL) 19 18 17 16 15

3. Mix the contents of the tube by gently vortexing and collect the reaction by brief

Note: The effi ciency of the second strand reaction is influenced by the amount and concen tra tion of the reactants, so the instructions must be followed as described for best results.

Overview

3

17

centrifugation. Place the tube at 37°C for 2 min to equilibrate the temperature.4. Add SuperScript® II RT according to the following table:

µg of mRNA (from step 1)

≤1 2 3 4 5

SuperScript® II RT (µL) 1 2 3 4 5

Mix gently and incubate at 37°C for 1 hour. Regardless of the amount of starting mRNA, the total volume should now be 20 µL.

Final composition of the reaction:50 mM Tris-HCl (pH 8.3)75 mM KCl3 mM MgCl210 mM DTT500 µM each dATP, dCTP, dGTP, and dTTP50 µg/mL Not I primer-adapter≤5 µg (≤250 µg/mL) mRNA10,000 to 50,000 units/mL SuperScript® II RT

5. Place the tube on ice to terminate the reaction.6. Remove 2 µL from the reaction, and add it to a microcentrifuge tube containing

43 µL of 20 mM EDTA (pH 7.5) and 5 µL of yeast tRNA. This mixture will be used in calculating first strand yield.

7. Take the remaining 18 µL of the first strand reaction, and continue immediately with the first two steps of the second strand reaction as described in Section 3.4.

8. While the second strand reaction is incubating, spot duplicate 10-µL aliquots from the diluted sample from step 6 of this protocol onto glass fiber filters. Dry one of the filters under a heat lamp or at room temperature. This filter will be used to determine the specific activity of the dCTP reaction.

9. Wash the other filter three times in sequence, for 5 minutes each time, in a beaker containing 50 mL of fresh, ice-cold 10% (w/v) TCA containing 1% (w/v) sodium pyrophosphate. Wash the filter once with 50 mL of 95% ethanol at room temperature for 2 minutes. Dry the filter under a heat lamp or at room temperature. This filter will be used to determine the yield of first strand cDNA.

10. Count both filters in standard scintillant to determine the amount of 32P in the reaction, as well as the amount of 32P that was incorporated. See Section 3.11 for information needed to convert the data into yield of first strand cDNA.

11. Precipitate the remaining 30 µL from step 6 of this protocol by adding 15 µL of 7.5 M NH4OAc, followed by 90 µL of absolute ethanol (–20°C). Vortex themixture thoroughly, and immediately centrifuge at room temperature for 20 minutes at 14,000 × g.

12. Remove the supernatant carefully, and overlay the pellet with 0.5 mL of 70% ethanol (–20°C). Centrifuge for 2 minutes at 14,000 × g, and remove the supernatant. Caution: If the first strand cDNA was labeled, the supernatant(s) will be radioactive. Dispose of this material properly.

13. Dry the cDNA at 37°C for 10 minutes to evaporate residual ethanol, and proceed to Section 3.11.

18

3.4 Second Strand SynthesisThis protocol is suitable for synthesizing second strand cDNA from ≤5 µg mRNA originally in the 20-µL first strand reaction.1. On ice, add the following reagents in the order shown to the first strand

reaction tube:Component ......................................................... Volume (µL)DEPC-treated water .............................................................935X Second Strand Buffer ......................................................3010 mM dNTP Mix ....................................................................3E. coli DNA Ligase (10 units/µL) .............................................1E. coli DNA Polymerase I (10 units/µL) ..................................4E. coli RNase H (2 units/µL) ...................................................1Final volume .......................................................................150

Final composition of the reaction:25 mM Tris-HCl (pH 7.5)100 mM KCl5 mM MgCl210 mM (NH4)2SO4

0.15 mM ß-NAD+

250 µM each dATP, dCTP, dGTP, dTTP1.2 mM DTT65 units/mL DNA ligase250 units/mL DNA polymerase I13 units/mL RNase H

2. Vortex the tube gently to mix, and incubate the completed reaction for 2 hours at 16°C. Do not let the temperature rise.

3. Add 2 µL (10 units) of T4 DNA polymerase, and continue incubating at 16°C for 5 min.

4. Place the reaction on ice, and add 10 µL of 0.5 M EDTA. 5. Add 150 µL of phenol:chloroform:isoamyl alcohol (25:24:1), vortex thoroughly,

and centrifuge at room temperature for 5 minutes at 14,000 × g to separate the phases. Carefully remove 140 µL of the upper, aqueous layer and transfer it to a fresh 1.5-mL microcentrifuge tube.

6. Add 70 µL of 7.5 M NH4OAc,followedby0.5mLofabsoluteethanol(–20°C).Vortex the mixture thoroughly, and immediately centrifuge at room temperature for 20 minutes at 14,000 × g.

7. Remove the supernatant carefully, and overlay the pellet with 0.5 mL of 70% ethanol (–20°C). Centrifuge for 2 minutes at 14,000 × g, and remove the supernatant. Caution: If the first or second strand cDNA was labeled, the supernatant(s) will be radioactive. Dispose of this material properly.

8. Dry the cDNA at 37°C for 10 minutes to evaporate residual ethanol, and proceed to Section 3.5.

3.5 Sal I Adapter Addition1. Add the following reagents on ice, in the order shown, to the cDNA from step 8

of Section 3.4.Component ......................................................... Volume (µL)DEPC-treated water .............................................................255X T4 DNA Ligase Buffer .....................................................10Sal I Adapters .......................................................................10T4 DNA Ligase .......................................................................5Final volume .........................................................................50

Note: If [α-32P]dCTP was added to the second strand reaction, remove 10 µL from the reaction, and add it to a microcentrifuge tube containing 35 µL of 20 mM EDTA (pH 7.5) and 5 µL of yeast tRNA. This mixture will be used in calculating second strand yield. Then proceed as described in steps 8 to 10 in Section 3.3 for processing and counting the filters.

Methods

3

19

Final composition of the reaction:50 mM Tris-HCl (pH 7.6)10 mM MgCl21 mM ATP5% (w/v) PEG 80001 mM DTT200 µg/mL Sal I Adapters100 units/mL T4 DNA Ligase

2. Mix gently, and incubate the reaction at 16°C for a minimum of 16 hours. For greatest convenience, simply let the reaction proceed overnight. Stop.

3. Add 50 µL of phenol:chloroform:isoamyl alcohol (25:24:1), vortex thoroughly, and centrifuge at room temperature for 5 minutes at 14,000 × g to separate the phases. Carefully remove 45 µL of the upper, aqueous layer and transfer it to a fresh 1.5-mL microcentrifuge tube.

4. Add 25 µL of 7.5 M NH4OAc,followedby150µLofabsoluteethanol(–20°C).Vortex the mixture thoroughly, and immediately centrifuge at room temperature for 20 minutes at 14,000 × g.

5. Remove the supernatant carefully, and overlay the pellet with 0.5 mL of 70% ethanol (–20°C). Centrifuge for 2 minutes at 14,000 × g, and remove the supernatant. Caution: If the first or second strand cDNA was labeled, the supernatant(s) will be radioactive. Dispose of this material properly.

6. Dry the cDNA at 37°C for 10 minutes to evaporate residual ethanol and proceed to Section 3.6.

3.6 Not I Digestion1. Add the following reagents on ice, in the order shown, to the cDNA from step 6

of Section 3.5:Component ......................................................... Volume (µL)DEPC-treated water .............................................................41REact 3 Buffer ........................................................................5Not I ........................................................................................4Final volume .........................................................................50

Final composition of the reaction:50 mM Tris-HCl (pH 8.0)10 mM MgCl2100 mM NaCl1200 units/mL Not I

2. Mix gently, and incubate the reaction for 2 hours at 37°C.3. Add 50 µL of phenol:chloroform:isoamyl alcohol (25:24:1), vortex thoroughly,

and centrifuge at room temperature for 5 minutes at 14,000 × g to separate the phases. Carefully remove 45 µL of the upper, aqueous layer and transfer it to a fresh 1.5-mL microcentrifuge tube.

4. Add 25 µL of 7.5 M NH4OAc,followedby150µLofabsoluteethanol(–20°C).Vortex the mixture thoroughly and immediately centrifuge at room temperature for 20 minutes at 14,000 × g.

5. Remove the supernatant carefully, and overlay the pellet with 0.5 mL of 70% ethanol (–20°C). Centrifuge for 2 minutes at 14,000 × g, and remove the supernatant. Caution: If the first or second strand cDNA was labeled, the supernatant(s) will be radioactive. Dispose of this material properly.

6. Dry the cDNA at 37°C for 10 minutes to evaporate residual ethanol and proceed to Section 3.7.

20

3.7 Column ChromatographyThis procedure optimizes size fractionation of the cDNA and makes the cloning of larger inserts more probable. This procedure also ensures that residual adapters and the Not I fragment released by restriction digestion of the primer-adapter with this enzyme do not enter into the library. Failure to adhere to these instructions can compromise the quality of your cDNA library.1. Dissolve the cDNA from step 6 of Section 3.6 in 100 µL TEN buffer [10 mM

Tris-HCl (pH 7.5), 0.1 mM EDTA, 25 mM NaCl; autoclaved], and let the pellet hydrate on ice.

2. While the pellet is hydrating, place one of the columns in a support. Remove the top cap first, and then the bottom cap. Allow the excess liquid (20% ethanol) to drain.

3. Pipet 0.8 mL of TEN buffer onto the upper frit, and let it drain completely. Repeat this step three more times for a total of 3.2 mL. Each 0.8-mL wash will take approximately 15 minutes, but it is important to do all four washes to remove the 20% ethanol from the column.

4. Label 20 sterile microcentrifuge tubes from 1 to 20, and place them in a rack with tube 1 under the outlet of the column.

5. Add the entire cDNA sample from step 1 of this protocol to the center of the top frit and let it drain into the bed. Collect the effluent into tube 1.

6. Add 100 µL of TEN buffer to the column, and collect the effluent into tube 2. Note: Let the column drain completely (i.e., until it stops dripping) before the addition of each new 100-µL aliquot.

7. Beginning with the next 100-µL aliquot of TEN buffer, collect single-drop (~35 µL) fractions into individual tubes. Continue adding 100-µL aliquots of TEN buffer until you have collected a total of 18 drops into tubes 3 through 20, one drop per tube.

8. Using an automatic pipet, measure the volume in each tube; use a fresh tip for each fraction to avoid cross contamination. Record each value in column A of one of the following work tables, using the sample table as a guide. Cap each tube after the volume has been measured and recorded. Calculate the cumulative elution volume with the addition of each fraction, and record this value in column B.

9. Identify the fraction for which the value in column B is closest to, but not exceeding, 550 µL (corresponding to fraction 12 in the sample table). Draw a horizontal line across the table immediately below this fraction. Do not use any of the subsequent fractions for your cDNA library; remove them to a separate tube rack to avoid accidentally using them in the remainder of the protocol. Important: Fractions collected after the 550-µL cutoff point (corresponding to tubes 13 through 20 in the sample table) will contain smaller cDNAs, unligated adapters, and the primer-adapter fragment released from the cDNA by Not I digestion. Use of these fractions significantly increases the risk of cloning the small primer-adapter fragment; also, because unligated Sal I adapters can compete with cDNA for available vector, the overall cloning efficiency is reduced.

10. Place the remaining tubes in a scintillation counter, and obtain Cerenkov counts for each fraction. Count the entire sample in the tritium channel; do not add scintillation fluid to the tubes. In column C, record the counts corresponding to each fraction. Note: Cerenkov counts above background should appear after passage of 400 to 450 µL of buffer.

11. For each fraction in which the Cerenkov counts exceed background (corresponding to fractions 8 to 12 in the sample table), calculate the amount of cDNA, using equation 5 in Section 3.11. Record each cDNA amount in column D.

Note: The sample table contains data typical for the size fractionation protocol; actual data will vary. The sample is provided merely to i l lustrate the decision-making process for selecting cDNA for the vector ligation reaction.

Note: Do not add more than one cDNA reaction per column. Addition of multiple reactions will result in poor cloning efficiency.

Note: If the flow rate is noticeably slower than 20 min/mL, do not use the column. Additionally, if the drop size from the column is not ~25 to 35 µL, do not use the column. The integrity and resolution of the cDNA might be compromised.

Tip: When collecting fractions, wear gloves that have been rinsed with ethanol to reduce static. Also, position the tube 1 to 2 cm from the bottom of the column to avoid the effects of static on drop size.

Methods

3

21

12. Divide each cDNA amount in column D by the fraction volume given in column A to determine the cDNA concentration per fraction. Record this value in column E.

13. The vector ligation reaction in Section 3.8 requires 10 ng of cDNA in ≤14 µL of TEN buffer. Note: If constructing a library in pEXP-AD502, 30 ng of cDNA will be needed. Examine the data entered in columns D and E of your work table, and decide whether to pool and precipitate early fractions, or (if applicable), to use 10 ng of cDNA from a suitable fraction directly in the vector ligation reaction. Guidelines for making this decision are provided in Section 3.11. If you have decided to pool and ethanol-precipitate the selected fraction(s), continue with step 14 of this protocol; if not, proceed directly to Section 3.8. Note: If you are stopping here, store the tubes at 4°C overnight.Stop.

14. If cDNA from two or more fractions must be pooled to obtain the 10 ng of cDNA needed for the vector ligation reaction, uncap the first selected tube (corresponding to fraction 8 in the sample table), and add cDNA from each subsequent fraction until there is at least 10 ng of cDNA in the tube. Measure the volume, and add 5 µL of yeast tRNA to the tube.

15. Add 0.5 volumes of NH4OAc, followed by 2 volumes of absolute ethanol(–20°C). Vortex the mixture thoroughly, and immediately centrifuge at room temperature for 20 minutes at 14,000 × g.

16. Remove the supernatant carefully, and overlay the pellet with 0.5 mL of 70% ethanol (–20°C). Centrifuge for 2 minutes at 14,000 × g, and remove the supernatant.

17. Dry the cDNA at 37°C for 10 minutes to evaporate residual ethanol, and dissolve in 10 µL of TEN buffer. Proceed to Section 3.8. Note: If you are stopping here (see Section 3.2.3), store the tubes at –20°C overnight. Stop.

3.8 Ligation of cDNA to the VectorThis protocol is intended for use with cDNA at ≥0.7 ng/µL. Do not proceed with this protocol until you have made the appropriate decisions regarding the choice of fractions for use in the ligation reaction. For more information, see Section 3.11.4. Note: If using pEXP-AD502 Sal I-Not I-Cut vector for cDNA library construction, the components below should be modified to: 2 µL pEXP-AD502, Not I-Sal I-Cut (50 ng/µL), and 30 ng cDNA in a 20-µL reaction.1. Add the following to a sterile 1.5-mL microcentrifuge tube at room temperature:

Component ................................................................Amount5X T4 DNA ligase buffer ....................................................4 µLNot I-Sal I-Cut vector .........................................................1 µLcDNA (≥0.7 ng/µL) ...........................................................10 ngDEPC-treated water .....sufficient to bring the volume to 19 µL

2. Add 1 µL of T4 DNA ligase, and mix by pipetting. Final composition of the reaction:

50 mM Tris-HCl (pH 7.6)10 mM MgCl21 mM ATP5% (w/v) PEG 80001 mM DTT2.5 µg/mL plasmid DNA, Not I-Sal I-Cut0.5 µg/mL cDNA50 units/mL T4 DNA ligase

3. Let the reaction incubate for 3 hours at room temperature or overnight at 4°C.4. Following incubation, the cDNA will be ligated into the cloning vector and ready

for transformation. Proceed to Section 3.9 or 3.10, as appropriate.

Note: If a large number of independent clones are needed, this ligation reaction can be scaled up to 160 µL in one tube.

Note: Although 10 to 20 ng of cDNA generally saturates the vector, the amount of cDNA that yields the maximal number of clones may be higher (e.g., 40 ng). As much as 14 µL of cDNA in TEN buffer may be added to the ligation reaction.

Note: See section 2.7 to determine the appropriate plasmid to use.

22

Methods

1 95 95 25

2 97 192 30

3 34 226 32

4 30 256 20

5 35 291 29

6 33 324 32

7 34 358 43

8 34 392 125 3.3 0.1

9 36 428 625 16 0.44

10 34 462 1,196 32 0.94

11 34 496 1,740 46 1.4

12 34 530 1,523 40 1.2

13 34 564

14 30 594

15 33 627

16 35 662

17 32 694

18 36 730

19 34 764

20 35 799

No.

A

FractionVolume

(µL)

B

TotalVolume

(µL)

C

CerenkovCounts(CPM)

D

Amount of cDNA

(ng)

EConcen-tration of

cDNA(ng/µL)

Sample Experiment

No.

A

FractionVolume

(µL)

B

TotalVolume

(µL)

C

CerenkovCounts(CPM)

D

Amount of cDNA

(ng)

EConcen-tration of

cDNA(ng/µL)

Experiment 1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

3

23

No.

A

FractionVolume

(µL)

B

TotalVolume

(µL)

C

CerenkovCounts(CPM)

D

Amount of cDNA

(ng)

EConcen-tration of

cDNA(ng/µL)

Experiment 2

No.

A

FractionVolume

(µL)

B

TotalVolume

(µL)

C

CerenkovCounts(CPM)

D

Amount of cDNA

(ng)

EConcen-tration of

cDNA(ng/µL)

Experiment 3

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

24

3.9 Introduction of Ligated cDNA into E. coli by TransformationIf you are going to introduce the ligated cDNA by transformation into E. coli cells such as MAX Efficiency® DH5α™ or MAX Efficiency® DH10B™ Competent Cells, use the following protocol. If you are going to introduce the ligated cDNA by electroporation into cells such as ElectroMAX™ DH10B Cells, use Section 3.10. These products are not part of the SuperScript® Plasmid System; however, they may be ordered in tandem with this system using a single catalogue number, or as separate products. Please see Section 7 for additional information.1. Transform a 100-µL aliquot of competent cells with 5 µL (12.5 ng vector) of the

reaction from step 3 of Section 3.8. Do not dilute the ligation reaction before adding it to the competent cells; the transformation protocol is otherwise identical to that accompanying Gibco® BRL Competent Cells. Place the remainder of the ligation reaction at 4°C until the results of the transformation are known the next day. To verify the transformation efficiency of the competent cells, use both the control DNA and the protocol supplied with the cells.

2. Add1mLofS.O.C.Mediumtothetransformedcellsandincubateat37°Cfor1 hour with vigorous aeration.

3. Plate aliquots of the cells on LB or YT plates containing 100 µg/mL ampicillin. Plate the equivalent of 10.0, 1.0, and 0.1 µL, made by serial dilution into S.O.C.Medium.Incubatetheplatesovernightat37°Candstoretheremainingtransformed cells at 4°C overnight.

4. Count the colonies on each plate, and calculate the number of colonies that would result from plating the entire transformation mixture. Using the remainder of the ligation reaction, transform the appropriate amount of cells needed to generate the desired number of transformants, and plate them at a density suitable for screening (17). The remaining transformed cells that had been stored overnight (from step 3 of this protocol) can also be plated. Some loss of viability will occur upon storage, but it generally will not be significant.

3.10 Introduction of Ligated cDNA into E. coli by Electroporation1. Add 5 µL of yeast tRNA and 12.5 µL of 7.5 M NH4OActotheligationreaction

from step 3 of Section 3.8.2. Add 70 µL of absolute ethanol (–20°C). Vortex the mixture thoroughly, and

immediately centrifuge at room temperature for 20 minutes at 14,000 × g.3. Remove the supernatant carefully, and overlay the pellet with 0.5 mL of 70%

ethanol (–20°C). Centrifuge for 2 minutes at 14,000 × g and remove the supernatant. Caution: If the first or second strand cDNA was labeled, the supernatant(s) will be radioactive. Dispose of this material properly.

4. Dry the ligated cDNA at 37°C for 10 minutes to evaporate residual ethanol.5. Add 3 to 5 µL of sterile, distilled water to the dried pellet, vortex, and collect the

contents of the tube by brief centrifugation.6. Add 1 µL of the ligated cDNA to 20 to 25 µL of electrotransformable cells such

as ElectroMAX™ DH10B Cells, and electroporate. Note: Efficient transformation of ElectroMAX™ DH10B Cells requires a field strength of ~16.6 kV/cm and a pulse length of ~4 ms. If you are using the Cell-Porator®

Electroporation System in conjunction with the Cell-Porator® Voltage Booster to generate the conditions for electroporation, please consult the operating instructions for these apparatus.

7. Add1mLofS.O.C.mediumtotheelectroporatedcellsandincubatethemat37°C for 1 hour with vigorous aeration.

Note: For a 160-µL reaction volume, use 5 µL of yeast tRNA, 82.5 µL of 7.5 M NH4OAcand560µL of absolute ethanol. Wash the pellet twice with 0.5 mL of 70% ethanol, dry and dissolve in 24 µL of sterile, distilled water.

Note: For the BioRad Gene Pulser®, use 1 µL of ligated cDNA to 40 µL of Electro-MAX cells or use 2-3 µL of ligated cDNA to 80 µL of ElectroMAX™ cells. The optimal electroporation voltage for ElectroMAX™ cells in the Gene Pulser® is 2.5 kV in a 0.1-cm gap chamber at settings of 100 ohms and 25 µF.

Methods

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8. Plate portions of the cells on LB or YT plates containing 100 µg/mL ampicillin. Plate the equivalent of 1.0, 0.1, and 0.01 µL, made by serial dilution into LB or YT medium. Incubate the plates overnight at 37°C and store the remaining transformed cells at 4°C overnight.

9. Count the colonies on each plate, calculate the number of colonies that would result from plating the entire 1 mL of cells, and plate them at a density suitable for screening (17). Some loss of viability will occur upon storage, but it generally will not be significant.

10. If the number of colonies obtained in the preceding step is not sufficient for your needs, or if you wish to have a reserve supply of independent transformants, electroporate additional 1-µL aliquots of the ligation mixture, as described in steps 6 and 7 of this protocol. Preserve the transformed cells by adding 1 mL of a sterile solution of 60% (v/v) LB or TYN medium and 40% (v/v) glycerol, mixing, and storing at –70°C.

3.11 Analysis of cDNA Products3.11.1 First Strand YieldThe overall yield of the first strand reaction is calculated from the amount of acid-precipitable radioactivity determined as described in Section 3.3. In order to perform the calculation, you must first determine the specific activity (SA) of the radioisotope in the reaction. The specific activity is defined as the counts per minute (cpm) of an aliquot of the reaction divided by the quantity (in pmol) of the same nucleotide in the aliquot. For [α-32P]dCTP, the specific activity is given by the relationship:

cpm/10 µL SA (cpm/pmol dCTP) = [1] 200 pmol dCTP/10 µL

The amount of dCTP contributed by the radiolabeled material is insignificant relative to the unlabeled nucleotide and is ignored in equation 1.

Oncethespecificactivityisknown,theamountofcDNAinthefirststrandreactioncan be calculated from the amount of acid-precipitable radioactivity determined from the washed filter:

Amount of (cpm) × (50 µL/10 µL) × (20 µL/2 µL) × (4 pmol dNTP/pmol dCTP) = [2] cDNA (µg) (cpm/pmol dCTP) × (3,030 pmol dNTP/µg cDNA)

The correction in the numerator takes into account that, on the average, four nucleotides will be incorporated into the cDNA for every dCTP scored by this assay. The factor in the denominator is the amount of nucleotide that corresponds to 1 µg of single-stranded DNA.

Example: The unwashed filter gave 50,000 cpm when it was counted. The specific activity of the dCTP is given by equation 1:

50,000 cpm/10 µL SA (cpm/pmol dCTP) = 200 pmol dCTP/10 µL

= 250 cpm/pmol dCTP

If 2 µg of starting mRNA was used and the washed filter gave 1,800 cpm, then the amount of cDNA is calculated using equation 2:

Amount of (1,800 cpm) × (50 µL/10 µL) × (20 µL/2 µL) × (4 pmol dNTP/pmol dCTP) = cDNA (µg) (250 cpm/pmol dCTP) × (3,030 pmol dNTP/µg cDNA)

= 0.5 µg first strand cDNA

This amount of first strand cDNA would represent a 25% yield relative to the 2 µg of mRNA starting material.

26

3.11.2 Second Strand YieldThe overall yield of the second strand reaction is calculated from the amount of acid-precipitable radioactivity determined as described in Section 3.4. In order to perform the calculation, you must first determine the specific activity of the radioisotope in the reaction. The specific activity is defined as the counts per minute of an aliquot of the reaction divided by the quantity of the same nucleotide in the aliquot. For [α-32P]dCTP, the specific activity is given by the relationship:

cpm/10 µL SA (cpm/pmol dCTP) = [3] 500 pmol dCTP/10 µL

The amount of dCTP contributed by the radiolabeled material is insignificant relative to the unlabeled nucleotide and is ignored in equation 3.

Once the specific activity is known, the amount of cDNA in the second strandreaction can be calculated from the amount of acid-precipitable radioactivity determined from the washed filter:

Amount of (cpm) × (50 µL/10 µL) × (150 µL/10 µL) × (4 pmol dNTP/pmol dCTP) = [4] cDNA (µg) (cpm/pmol dCTP) × (3,030 pmol dNTP/µg cDNA)

The correction in the numerator takes into account that, on the average, four nucleotides will be incorporated into the cDNA for every dCTP scored by this assay. The factor in the denominator is the amount of nucleotide that corresponds to 1 µg of single-stranded DNA.

Example: The unwashed filter gave 300,000 cpm when it was counted. The specific activity of the dCTP is given by equation 3:

300,000 cpm/10 µL SA (cpm/pmol dCTP) = 500 pmol dCTP/10 µL

= 600 cpm/pmol dCTP

If 2 µg of starting mRNA was used and the washed filter gave 2,500 cpm, then the amount of cDNA is calculated using equation 4:

Amount of (2,500 cpm) × (50 µL/10 µL) × (150 µL/10 µL) × (4 pmol dNTP/pmol dCTP) = cDNA (µg) (600 cpm/pmol dCTP) × (3,030 pmol dNTP/µg cDNA)

= 0.4 µg second strand cDNA

3.11.3 Gel AnalysisThe first or second strand cDNA, if labeled with 32P, can be analyzed by alkaline agarose gel electrophoresis to estimate the size range of products synthesized (18). Gibco BRL Horizon® 11·14 Apparatus is ideally suited for analyzing small amounts of cDNA, although any standard electrophoresis apparatus can be used.

The ethanol-precipitated first strand sample is dissolved in 10 µL 1X alkaline agarosegelsamplebuffer [30mMNaOH,1mMEDTA,10%(v/v)glycerol,0.01%bromophenol blue].Other samples (such as theGibcoBRL 1KbDNA Ladder,labeled with 32P) can be electrophoresed after addition of a suitable volume of a more concentrated sample buffer; the only precaution is to chelate any Mg2+ by addition of EDTA prior to adding the alkaline sample buffer.

The gel [1.4% (w/v)] should be cast in the appropriate volume of 30 mM NaCl, 2 mM EDTA (alkaline buffer cannot be used because it will degrade the agarose when the solution is microwaved to melt the agarose), and should be equilibrated for 2–3 hours in alkaline electrophoresis buffer (30mMNaOH, 2mMEDTA) beforeloading the samples. Electrophoresis should be for 5–6 hours at 50 V or for 16–18 hours at 15 V. The gel should be dehydrated under vacuum until the buffer is removed, then under heat and vacuum for several hours to complete the drying. The dried gel should then be exposed to x-ray film overnight at room temperature.

Methods

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27

When a heterogeneous mRNA population is fractionated by alkaline gel electrophoresis, a continuum of fragments ranging in size from 500–5000 nucleotides makes up the bulk of the first and second strand cDNA. Figure 8 shows an alkaline electrophoretic analysis of 32P-labeled first and second strand cDNA synthesized from HeLa mRNA with the SuperScript® Plasmid System.

3.11.4 Analysis of cDNA from the cDNA Size Fractionation ColumnCalculation of the amount of size-fractionated cDNA in each column fraction is necessary to ensure that the proper decisions are made concerning fraction selection and that the cDNA is used economically in the ligation reaction. The Cerenkov counts are approximately 50% of the radioactivity that would be measured in scintillant. The counts are converted into nanograms of cDNA using the specific activity determined in Section 3.3 or 3.4. The amount of cDNA (as double strand) in each fraction is given by the following relationship:Amount of (Cerenkov cpm) × 2 × (4 pmol dNTP/pmol dCTP) × (1,000 ng/µg ds cDNA) = [5]ds cDNA (ng) SA (cpm/pmol dCTP) × (1,515 pmol dNTP/µg ds cDNA)

Example: If one of the fractions from the column gave 1,500 cpm when counted by Cerenkov radiation (first-strand-labeled), then the amount of cDNA in that fraction is calculated using equation 5:Amount of (1,500 cpm) × 2 × (4 pmol dNTP/pmol dCTP) × (1,000 ng/µg ds cDNA) = ds cDNA (ng) SA (cpm/pmol dCTP) × (1,515 pmol dNTP/µg ds cDNA)

= 32 ng

After calculating the amount and the concentration of cDNA in each fraction (columns D and E of the work table), you are ready to select and recover cDNA for use in the vector ligation reaction in Section 3.8. At this point, you have two options to consider:1. If you wish to maximize the average insert size in your cDNA library and your

earliest selected fraction (corresponding to fraction 8 in the sample table) contains <10 ng of cDNA, you will need to pool cDNA from this fraction with at least a portion of a subsequent fraction. Because this procedure will produce a solution of cDNA at <0.7 ng/µL (too dilute for use in the ligation reaction), you will also need to ethanol-precipitate the pooled cDNA. For example, fraction 8 from the sample table contains 3.3 ng of cDNA in 34 µL, and fraction 9 contains 16 ng of cDNA in 36 µL. If all of fraction 8 is combined with 15 µL (6.6 ng of cDNA) from fraction 9, the pool will contain 10 ng of cDNA in 47 µL, or 0.2 µg/µL. The ethanol precipitation steps in Section 3.7 will then bring the concentration to 1 ng/µL.

2. If any of the selected fractions contain ≥10 ng of cDNA at ≥0.7 ng/µL (as with fraction 10, 11, and 12 in the sample table in Section 3.7), you can use 10 ng from the fraction directly in the vector ligation reaction. Note: If this fraction is not the earliest selected, based on Cerenkov counts (corresponding to fraction 8 in the sample table), the average insert size in your cDNA library will be smaller than could be obtained through additional pooling and ethanol precipitation steps. Figure 9 provides an electrophoretic display of the size ranges of cDNA obtained in fractions 8 through 19 in the experiment that generated the sample table data.

Figure 8. Alkaline agarose gel analysis of first and second Strand cDNA synthes ized wi th the SuperScript® Plasmid System. Samples of 32P-labeled first or second strand cDNA made from HeLa mRNA were ethanol-precipitated, dissolved in alkaline agarose sample buffer, and electrophoresed on a 1.4% agarose gel at 15 V for 16 h.

Mar

kers

Firs

t Str

and

Seco

nd S

tran

d

kb

7.1 6.1 5.1

4.0

3.0

2.0

1.6

1.0

0.5

28

The preceding options are provided assuming that you wish to achieve maximum transformation efficiencies in keeping with the design of Section 3.8. There is a third option available: if you wish merely to maximize insert size and are willing to accept lower transformation efficiencies to attain this goal, you may use your earliest selected fraction in Section 3.8 even if it contains <10 ng of cDNA. However, you must ethanol-precipitate the fraction prior to using it in the vector ligation reaction.

Please note that this option should preferably be attempted using electroporation methods, which offer higher transformation efficiencies (and thus a greater chance of generating a sufficiently large cDNA library).

Figure 9. Electrophoretic analysis of size-fractionated cDNA. [32P]cDNA was fractionated on a 1-mL prepacked column equilibrated in TEN buffer. Single-drop fractions (~35 µL each) were collected, and aliquots were analyzed by electrophoresis on a 1% agarose gel in 40 mM Tris-acetate (pH 8.3), 5 mM sodium acetate, 1 mM EDTA. The gel was electrophoresed at 200 V for 2 h.

Column Fraction

8 9 10 11 12 13 14 15 16 17 18kb

12.2

7.1

5.1

3.0

2.0

1.6

1.0

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Troubleshooting Guide

4.1 Isolation of mRNAIn the first step of cDNA library construction, RT converts the sequence information of the mRNA to first strand cDNA. The quality of the mRNA used as the template will influence profoundly the yield and size distribution of the first strand product. We recommend a guanidine isothiocyanate-based homogenization procedure to ensure rapid inactivation of RNases and general deproteinization, followed by two selections of mRNA by oligo(dT) cellulose chromatography to enrich maximally for poly(A)+ RNA.

The mRNA preparation can be analyzed by formaldehyde agarose gel electrophoresis (19) and ethidium bromide staining (20). The gel should reveal a smear of fluorescent material, and perhaps some discrete fragments corresponding to abundant mRNAs. Residual 18S or 28S rRNAs (approximately 2,000 or 5,000 bases in length, respectively, in mammalian cells) will be visible even in mRNA preparations highly enriched for poly(A)+ RNA, and will be indicative of an intact mRNA population. If neither rRNA is visible and the distribution of the mRNA is not centered in the 1- to 3-kb range, then you will need to consider troubleshooting your RNA isolation procedure. Examples of representative mRNA preparations have been published (21).

4.2 First Strand ReactionThe conditions described for the first strand reaction have been optimized; thus, it is imperative to follow Section 3.3 explicitly. Do not increase the size of the first strand reaction from 20 µL.

First strand yields will vary widely; we routinely obtain 25% to 35% yields from HeLa mRNA preparations using 2 µg of mRNA in the first strand reaction. Analysis of the products by alkaline gel electrophoresis reveals a distribution from 0.5 to >7 kb (see figure 6). A lower yield does not necessarily indicate that a library cannot be made, so long as the size distribution of the products is consistent with the size distribution of the mRNA. A lower distribution suggests RNase contamination. If this occurs, use the control RNA to synthesize first strand cDNA and examine the products by alkaline gel electrophoresis.

If you obtain a poor yield in the first strand reaction, the control RNA can be used to verify that the system components are working properly. This RNA is 2.0 kb long and can be substituted directly into the first strand reaction as described in Section 3.3—use of the extra components provided (see Utilization of Reagents at the beginning of Section 3) allows you to reverse transcribe the control RNA without losing the capability to construct three cDNA libraries as described in Sections 3.3 through 3.10. The first strand yield using the control RNA is generally 40% to 50%. If desired, the control RNA can be taken through the entire procedure and cloned into the Not I-Sal I-Cut vector.

4.3 Second Strand Reaction The second strand reaction in Section 3.4 is not sensitive to RNase contamination, but strict adherence to good laboratory practice is still required. The second strand reaction is generally efficient, and yields of 80% to 100% (relative to the amount of first strand cDNA synthesized) are common. The distribution of second strand cDNA products should look like the distribution of the first strand cDNA products when analyzed by alkaline agarose gel electrophoresis (see Figure 8).

Note: TRIzol® Reagent can be used to isolate high quality total RNA from cells and tissue.

Blue-white screening can be performed withpSPORT1.

30

Troubleshooting Guide

Unlike some second strand procedures that do not require E. coli RNase H (10), this enzyme must be included to provide initiation points for nick translation by DNA polymerase I when first strand cDNA is synthesized by SuperScript® RT. Furthermore, the reaction must be incubated at ≤16°C to prevent spurious synthesis by DNA polymerase I, although this is contrary to the original descriptions in the literature (8,9).

Dilution of the first strand reaction precisely as specified in Section 3.3 is extremely important because the pH of the second strand reaction differs from that of the first strand reaction. This pH change influences the activity of the 3´→5´ and 5´→3´ exonucleases of DNA polymerase I, thereby limiting the number of nucleotides that are removed from the termini of the cDNA as the second strands are completed.

4.4 Sal I Adapter Addition and Not I Digestion These protocols are the most difficult to troubleshoot because they will not be suspect until the cloning efficiency is determined. The adapter addition step is assayed most readily by nondenaturing polyacrylamide gel electrophoresis. If a 10-µL aliquot of the adapter ligation reaction is electrophoresed on a 12% acrylamide gel and the DNA is visualized by ethidium bromide staining, the ligation of two adapters to each other at their blunt ends will be evidenced by a conspicuous 32-bp fragment. Although this does not verify that the adapters have ligated to the cDNA, it does show that the ligation reaction in Section 3.5 is functionally sound.

The digestion conditions in Section 3.6 provide for a large excess of Not I (45 units to digest ≤2 µg of cDNA, assuming a 40% first strand yield from 5 µg of mRNA and no other loss of material through manipulations) and an extended incubation to ensure complete digestion.

4.5 Column Chromatography The most likely problems with Section 3.7 will arise either from running the column too quickly—it is imperative to let it go dry between 100-µL aliquots—or taking fractions beyond 550 µL. If fractions beyond 550 µL are used, the library will contain more small cDNAs (100 to 500 bp) or apparently “empty” clones. The empty clones arise from ligation of the small (44-bp) fragment released by cleavage of the Not I site in the primer-adapter. Please see Section 3.11.4 and the sample table in Section 3.7 for representative size fractionation results and for guidelines on selecting the proper fractions.

4.6 Ligation of cDNA to the VectorIf the transformation efficiency is low (<106 transformants/µg of cDNA, assuming that supercoiled plasmid yields 108 transformants/µg), the ligation reaction may not be proceeding properly. If the remainder of the ligation reaction is electrophoresed on an agarose gel (along with 1 µL of the vector as a control, run in an adjacent lane) and visualized by ethidium bromide staining (using a strong 254-nm UV transilluminator), the DNA should smear upward from the position of the 4.1-kb vector. If the DNAs did not ligate, repeat Section 3.8 using a smaller volume (for example, 2 µL) of the column fraction containing the highest concentration of cDNA; however, do not go beyond the first five fractions that contain cDNA. This procedure will minimize inhibition if the column buffer is a problem, although up to 14 µL of the column buffer generally can be used in the ligation.

Another cause of low transformation efficiencies is using an insufficient amount of cDNA in the ligation reaction. Recheck all calculations used to generate the data for the table, to rule out the possibility of a simple mathematical error. Also, if you chose not to ethanol-precipitate the cDNA, when it would have been the recommended procedure, following size fractionation, return to step 13 of Section 3.7 to ethanol-precipitate the cDNA, and proceed to Section 3.8.

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31

4.7 Introduction of Ligated cDNA into E. coliThe most commonly used method for introducing the ligated cDNA into E. coli is to transform competent cells. We have routinely used MAX Efficiency® DH5α and MAX Efficiency® DH10B™ Competent Cells as hosts for cDNA libraries. The number of transformants that we obtain per 0.1-mL transformation with these cells ranges from 0.4 to 1.4 × 105 transformants, when the ligation reaction and transformation conditions described in Sections 3.8 and 3.9 are followed. We have optimized these conditions to produce the greatest number of transformants per transformation using the smallest amount of cDNA possible.

While it may be possible to increase the number of transformants per µg of cDNA by altering the ligation and/or transformation conditions, the clonal output per transformationdropssharply.Ofcourse, ifyouchoosetouseyourowncompetentcells for establishing the library, we strongly recommend that you optimize the ligation reaction and transformation parameters.

If you choose to introduce the ligated cDNA into E. coli by electroporation, the DNA in the ligation reaction must be concentrated by ethanol precipitation and dissolved in distilled water, as described in Section 3.10. This step is necessary due to the high electric potentials generated during the electroporation procedure. We obtain approximately 1.0 × 106 transformants from electroporation of ElectroMAX™ DH10B™ Cells (see Section 3.10).

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Additional Protocols

5.1 Expansion of Plasmid cDNA LibrariesSemi-solid amplification of primary cDNA transformants minimizes representational biases that can occur during the expansion of plasmid cDNA libraries (25). Life Technologies has further modified this protocol such that the amplification is done at 30°C, helping to stabilize unstable clones (26).

Caution: Bottles of semi-solid agar containing suspended colonies must be handled gently and incubated without disturbance. Rough handling or bumping of the incubator will cause micro colonies to fall out of solution. Incubators that have fans can cause colonies to fall out of solution.1. Prepare 2.0 L of 2X LB. Remove 175 mL of the 2X LB to make the 2X LB

Glycerol.2. Place a large stir bar and 1.35 g SeaPrep® agarose into each of four 500-mL

autoclavable bottles. Place bottles on stir plates. With the stir plate turned on, add 450 mL of 2X LB to each bottle (avoid the formation of large clumps of agarose).

3. Autoclave these bottles of 2X LB agarose for 30 minutes.4. Cool bottles in 37°C water bath for ~2 hours until the medium reaches 37°C.5. After the medium reaches 37°C, add carbenicillin to 50 µg/mL (preferred

antibiotic) or add ampicillin to 200 µg/mL. Mix on stir plate.6. To each bottle add 4 × 105 to 6 × 105 primary cDNA transformants (colonies

from original library), and mix thoroughly on a stir plate for 2 minutes. Tighten caps.

7. Place bottles in an ice-water bath (0°C) such that the water in the bath is at the same level as the medium in the bottle. Incubate for 1 hour in the ice bath.

8. Gently remove bottles from the ice bath and wipe the water off the outside of the bottles. Loosen bottle caps. Place these bottles in a gravity flow incubator set at 30°C.

9. Incubate these bottles for 40–45 hours without disturbance. Place a centrifuge rotor at room temperature the morning of harvest.

10. Pour contents of bottles into centrifuge bottles and centrifuge at 10,400 × g for 20 minutes at room temperature

11. Pour off the supernatant.12. Resuspend the cells in a total of 100 mL 2X LB Glycerol (12.5%). Remove two

100-µL aliquots for plating, further analysis, and colony estimate. Cells can be filtered through sterile cheesecloth to remove agarose clumps if present.

13. Subdivide the cells into ~10-mL aliquots and store at -70°C. It is also useful to make a number of 1-mL and 100-µL aliquots.

14. Frozen cells can be used to prepare DNA for plating to screen or can be further amplified in liquid at 30°C to obtain DNA. Use 2.5 × 109 cells/100 mL growth medium for further expansion of the library.

Note: Carbenicillin reduces satellite formation.

Caution: Make sure that the rotor was set at room temperature for at least 2 hours before adding the bottles. Rotors at 4°C will cause solidification of agar.

5

33

Media2X LBComponent Amount/literBacto-Tryptone ................................................................. 20 gBacto-Yeast Extract .......................................................... 10 gNaCl .................................................................................. 10 g

2X LB Glycerol (12.5%)Component Amount/200 mL2X LB ........................................................................... 175 mLGlycerol (100%) ..............................................................25 mL

Filter-sterilize and store for up to 2 months at room temperature.

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34

References

1. Travis, G.H. and Sutcliffe, J.G. (1988) Proc. Natl. Acad. Sci. USA 85, 1696.2. Aviv, H. and Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, 1408.3. Kotewicz, M.L., D’Alessio, J.M., Driftmeir, K.M., Blodgett, K.P., and Gerard,

G.F. (1985) Gene 35, 249.4. Kotewicz, M.L., Sampson, C.M., D’Alessio, J.M., and Gerard, G.F. (1988)

Nucl. Acids Res. 16, 265.5. Berger, S.L., Wallace, D.M., Puskas, R.S., and Eschenfeldt, W.H. (1983)

Biochemistry 22, 2365.6. Gerard, G.F., D’Alessio, J.M., and Kotewicz, M.L. (1989) Focus® 11, 66.7. D’Alessio, J.M., Gruber, C.E., Cain, C., and Noon, M.C. (1990) Focus 12, 47.8. Okayama,H.andBerg,P.(1982)Mol. Cell. Biol. 2, 161.9. Gubler, U. and Hoffman, B.J. (1983) Gene 25, 263.10. D’Alessio, J.M. and Gerard, G.F. (1988) Nucl. Acids Res. 16, 1999.11. Maniatis, T., Fritsch, E.F., and Sambrook, J. (1989) Molecular Cloning: A

Laboratory Manual, (2nd Ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

12. Henikoff, S. (1984) Gene 28, 351.13. Vieira, J. and Messing, J. (1987) Methods Enzymol. 153, 3.14. Kunkel,T.A.,Roberts,J.D.,andZakour,R.A.(1987)Methods Enzymol. 154,

367.15. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979)

Biochemistry 18, 5294.16. Han, J.H., Stratowa, C., and Rutter, W.J. (1987) Biochemistry 26, 1617.17. Hanahan, D. and Meselson, M. (1983) Methods Enzymol. 100, 333.18. McDonnell, M.W., Simon, M.N., and Studier, F.W. (1977) J. Mol. Biol. 110,

119.19. Gerard, G.F. and Miller, K. (1986) Focus 8:3, 5.20. Matathias, A.S. and Komro, C. (1989) Focus 11, 79.21. D’Alessio, J.M. and Noon, M.C. (1989) Focus 11, 49.22. Gerard, G.F., Schmidt, B.J., Kotewicz, M.L., and Campbell, J.H. (1992) Focus

14, 91.23. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156.24. Simms, D. (1995) Focus 17, 39.25. Kriegler, M. (1990) Gene Transfer and Expression: A Laboratory Manual,

Stockton Press, New York.26. Hanahan, D., Jessee, J. and Bloom, F.R. (1991) Methods Enzymol 204, 63.

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Related Products

Product Size Cat. No

Combination Systems:SuperScript® Plasmid System and one set 19625-011 ElectroMAX™ DH10B™ Competent Cells

SuperScript® cDNA Libraries:Visit www.lifetechnologies.com for a complete product listing.

Products for RNA PurificationFastTrack® 2.0 mRNA Isolation Kit 6 rxns K1593-02 3 x 6 rxns K1593-03TRIzol® Reagent 100 mL 15596-026 200 mL 15596-018TRIzol® LS Reagent 100 mL 10296-010 200 mL 10296-028UltraPure™ Guanidine Hydrochloride 500 g 15502-016Guanidine Isothiocyanate 500 g 15535-016

UltraPure™ DEPC-Treated Water 4 × 1.25 mL 10813-012UltraPure™ Phenol 500 g 15509-037UltraPure™ Phenol:Chloroform:Isoamyl Alcohol, (25:24:1, v/v/v) 100 mL 15593-031 RNaseOUT™RecombinantRibonucleaseInhibitor 5,000units 10777-019RNase AWAY™ Reagent 250 mL 10328-0110.5-10 Kb RNA Ladder 75 µg 15623-200

Products for TransformationMAX Efficiency® DH5α™ Competent Cells 1 mL 18258-012MAX Efficiency® DH10B™ Competent Cells 1 mL 18297-010IPTG 1 g 15529-019S.O.C.Medium 10× 10 mL 15544-034X-gal 100 mg 15520-034Ampicillin, Sodium Salt, lyophilized 20 mL 11593-019Carbenicillin, Disodium salt 5 g 10177-012

Products for ElectroporationElectroMAX™ DH10B™ Cells 5 × 0.1 mL 18290-015ElectroMAX™ DH12S™ Cells 5 × 0.1 mL 18312-017ElectroMAX™ Stbl4™ Cells 5 × 0.1 mL 11635-018

Other Related ProductsBaculovirus Expression System with Gateway® Technology 20 reactions 11827-011Gateway® Vector Conversion System 20 reactions 11828-029SuperScript® II Reverse Transcriptase 10,000 units 18064-014SuperScript® Choice System for cDNA Synthesis 3 reactions 18090-019Second Strand Buffer 0.5 mL 10812-014Oligo(dT)12-18 Primer 25 µg 18418-012UltraPure™ Glycogen 100 µL 10814-010cDNA Size Fractionation Columns 3 columns 18092-0151 Kb Plus DNA Ladder 250 µg 10787-018 1 mg 10787-026UltraPure™ Agarose 100 g 15510-019 500 g 15510-027UltraPure™ 10X TBE Buffer 1 L 15581-044 10 L 15581-028

For information on our Gateway® Cloning a n d E x p r e s s i o n S e r v i c e s , v i s i t www.lifetechnologies.com.

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Notes

37

Notes

Notes

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13 August 2015