micro-fasttrack 2.0 kit - thermo fisher...

Micro-FastTrack™ 2.0 Kit For isolation of mRNA from small samples

Catalog Number K1520-02

Part Number 25-0023 Publication Number MAN0000007 Revision 3.0

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

USER GUIDE

3

Contents

Kit Contents and Storage .................................................................................................................................... 4

Introduction ................................................................................................................... 5 Product Overview ............................................................................................................................................... 5

Methods ......................................................................................................................... 7 Preparing Samples – General ............................................................................................................................. 7

Preparing Samples – Tissue Culture Cells ....................................................................................................... 8

Preparing Samples – Fresh and Frozen Tissue ................................................................................................ 9

Preparing Samples – Plants and Total RNA .................................................................................................. 10

Basic mRNA Isolation Method ........................................................................................................................ 11

Troubleshooting ................................................................................................................................................. 16

Appendix ...................................................................................................................... 17 RT-PCR Protocol ................................................................................................................................................ 17

Accessory Products ........................................................................................................................................... 20

Technical Support .............................................................................................................................................. 21

References ........................................................................................................................................................... 22

4

Kit Contents and Storage

Shipping and Storage

The Micro-FastTrack™ 2.0 mRNA Isolation Kit is shipped at room temperature and may be stored at room temperature for up to 6 months after receipt of the kit. Note that oligo(dT) cellulose is hygroscopic and sensitive to light. Upon receipt of the kit, remove 20 tubes of the premeasured oligo (dT) cellulose powder and store in a dessicator protected from light at room temperature.

For long-term storage (>6 months), store the oligo(dT) cellulose at –20°C in a dessicator protected from light. The Proteinase K solution is stable for 1 year when stored at room temperature. For long-term storage (>1 year) or if room temperature is >25ºC, store the Proteinase K solution at 4ºC.

Kit Contents The Micro-FastTrack™ 2.0 Kit contents are listed in the following table. Sufficientreagents are supplied to perform 20 mRNA isolations.

Item Volume Composition

Stock Buffer 20 mL 200 mM NaCl 200 mM Tris-HCl, pH 7.5 1.5 mM MgCl2 2% SDS

Proteinase K, 20 mg/mL in storage buffer

425 μL Proprietary

Binding Buffer 4 × 38 mL (152 mL)

500 mM NaCl 10 mM Tris-HCl, pH 7.5 in DEPC-treated water

Low Salt Wash Buffer 8 mL 250 mM NaCl 10 mM Tris-HCl, pH 7.5 in DEPC-treated water

Elution Buffer 8 mL 10 mM Tris-HCl, pH 7.5 in DEPC-treated water

2 M Sodium Acetate 1 mL 2 M Sodium Acetate, pH 5.2 in DEPC-treated water

5 M NaCl 1.3 mL 5 M NaCl in DEPC-treated water

Glycogen Carrier 200 μL 2 mg/mL in DEPC-treated water

Pre-measured Oligo(dT) 20-30 Cellulose Powder

20 × 25 mg

Lyophilized oligo(dT) cellulose

Disposable Spin Columns with microcentrifuge tubes

20 each —

Microcentrifuge Tubes 20 each —

Micro-FastTrack™ 2.0 Quick Reference Card

Also included in this kit is a Quick Reference Card that can be used as a checklist. Each step can be conveniently marked with a laboratory marker to keep track of centrifugations, transfers, and washes. Marks may be removed with ethanol before next use.

5

Introduction Product Overview

Description of the System

The Micro-FastTrack™ 2.0 mRNA Isolation Kit allows isolation of polyA+ RNA directly from cells (1 × 102–5 × 106), tissue (10–200 mg), or total RNA (100–500 μg) in 2–3 hours using minimal equipment (a water bath, a microcentrifuge, and a syringe fitted with an 18-21 gauge needle) without the need for ultracentrifugation or guanidinium lysis.

Typical yields The Micro-FastTrack™ 2.0 mRNA Isolation Kit typically yields the following

amounts of mRNA:

Sample Starting Amount mRNA Yield

Cultured mammalian cells (e.g., HeLa, BHK) 5 × 106 cells 1–3 μg

Cultured insect cells 5 × 106 cells 3–5 μg

Human fetal heart 200 mg tissue 8–12 μg

Yields of mRNA vary depending on the type of tissue and the stage of differentiation of your cells. The yield of polyA+ RNA from total RNA should be approximately 1–5% of the amount of starting material. PolyA+ RNA isolated with the Micro-FastTrack™ 2.0 Kit is suitable for RACE, microarray analysis, RT-PCR, and northern blots (see page 20 for ordering information).

Flow Chart of Process

The following figure outlines the Micro-FastTrack™ 2.0 mRNA Isolation process.

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6

Product Overview, Continued

Materials Needed Solutions

• Phosphate-Buffered Saline (PBS) to wash tissue culture cells (see page 8)

• 100% ethanol

• 70% ethanol (in DEPC-treated water)

Equipment

• Sterile 15-mL conical centrifuge tubes (cultured cells)

• Water baths (45°C, 65°C)

• Liquid nitrogen (tissues)

• Sterile knife (tissues)

• Sterile 1-cc syringes with 18–21 gauge needles

• Table-top centrifuge

• Microcentrifuge

• Rocking platform or rotating wheel

• Mortar, pestle, and 1 mL Dounce Homogenizer (tissues optional)

7

Methods

Preparing Samples – General

Introduction Review this section carefully. The method of sample preparation depends on whether your sample is tissue culture cells, tissues (including plants), or total RNA. The lysate or total RNA solution you produce in this section is then used directly for mRNA isolation. Familiarize yourself with the basic mRNA isolation protocol on page 11. Review the protocols carefully before starting your experiment. Once you lyse your sample, do not stop until the mRNA is in ethanol (see Elution and Precipitation of the mRNA, Step 6, page 14). The entire procedure should take approximately 2–3 hours depending on the nature of your starting material.

General Handling of RNA

When working with RNA:• Use disposable, individually wrapped, sterile plasticware

• Use only sterile, new pipette tips and microcentrifuge tubes

• All plasticware provided in the Micro-FastTrack™ 2.0 Kit is RNase-free

• Glassware used for RNA work should be cleaned with detergent, thoroughly rinsed, autoclaved, and then oven baked at >210°C for at least three hours before use. Oven baking inactivates ribonucleases and ensures that no other nucleic acids (such as plasmid DNA) are left on the surface of the glassware.

• Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin.

• Always use proper microbiological aseptic technique when working with RNA.

You may use RNase AWAY® Reagent, a non-toxic solution available from Life Technologies (see page 20) to remove RNase contamination from surfaces. For further information on controlling RNase contamination, see Ausubel, et al., 1994, or Sambrook, et al., 1989.

Preparing Lysis Buffer

You need to prepare the Micro-FastTrack™ 2.0 Lysis Buffer immediately before use. Follow the instructions below to prepare the Lysis Buffer.

1. Check the Stock Buffer for a white precipitate (SDS). If the Stock Buffer has a white precipitate, heat the solution to 65°C until dissolved. Cool the solution to room temperature prior to adding the Protein/RNase Degrader.

2. Add 20 μL of Proteinase K to 1 mL of Stock Buffer for each intended isolation. Use immediately.

8

Preparing Samples – Tissue Culture Cells

Preparing Tissue Cultured Cells

We recommend using 1 × 102–5 × 106 cells for each mRNA isolation. Remember to prepare your Micro-FastTrack™ 2.0 Lysis Buffer immediately before use (see page 7).

1. Wash the cells in 4°C phosphate buffered saline (PBS) solution (available from Life Technologies, visit www.lifetechnologies.com for details).

2. Transfer the cells to a 15-mL sterile, conical centrifuge tube. Centrifuge the cells and resuspend the pellet in 1 mL of PBS.

3. Transfer the cells to a sterile microcentrifuge tube. Centrifuge the cells and continue with Step 4, or flash freeze the pellet in liquid nitrogen and store at –80°C.

4. Resuspend the cell pellet in 1 mL of Micro-FastTrack™ 2.0 Lysis Buffer containing Proteinase K (see page 7) to lyse the cells. If the pellet was frozen and does not thaw quickly, place the tube with the cells and buffer in a 45°C water bath for 1–2 minutes. Vortex to completely resuspend the cells.

5. Pass the lysate 2–4 times through a sterile plastic 1-mL syringe fitted with a 18–21 gauge needle. If the lysate is still viscous, continue to pass the solution through the syringe until it is no longer viscous (≤10 times). Proceed to the Basic mRNA Isolation Method, Step 1, page 11.

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9

Preparing Samples – Fresh and Frozen Tissue

Preparing Tissue for Storage

To achieve high yields of mRNA from plant, insect, or mammalian tissues, ~10–200 mg of tissue per isolation is recommended. Perform all lysis and washing steps as described in the protocols to ensure consistently high yields.

1. Quickly excise the tissue, cut into 0.5 cm2 pieces with a sterile knife or razor blade, and immediately freeze in liquid nitrogen.

2. Transfer tissue pieces to a sterile cryovial with a screw-cap and store the tissue at –80°C. Frozen tissue is stable at –80°C for over 1 year. Proceed to Preparing Fresh or Frozen Tissue.

Preparing Fresh or Frozen Tissue

It is important that frozen tissue is thawed and homogenized in the presence of lysis buffer. This ensures immediate inactivation of any RNases that are released as the cells lyse. Complete homogenization is critical for complete cell lysis and inactivation of RNases.

Before beginning, have the following materials on hand:

• Micro-FastTrack™ 2.0 Lysis Buffer (see page 7), preheated to 45°C

• Sterile 1-mL syringe fitted with a 18–21 gauge needle or a mortar, pestle, and a 1-mL Dounce homogenizer

• Sterile microcentrifuge tubes

Procedure

1. Place ~200 mg fresh or frozen tissue in a preweighed, sterile, microcentrifuge tube and weigh. If the tissue is frozen, allow any remaining liquid nitrogen to evaporate. Add 1 mL Micro-FastTrack™ 2.0 Lysis Buffer containing Proteinase K, preheated to 45°C.

2. Homogenize the tissue by passing through a syringe fitted with a 18–21 gauge needle. It may be necessary to break up the tissue by grinding it against the side of the tube with the needle. Continue to work the tissue in this manner until the entire suspension will draw up into the syringe.

Optional: If the tissue is difficult to homogenize with the syringe and needle, you may use a mortar and pestle or a 1-mL Dounce homogenizer.

3. Proceed to the Basic mRNA Isolation Method, Step 1, page 11.

10

Preparing Samples – Plants and Total RNA

Reminder Remember to prepare the Micro-FastTrack™ 2.0 Lysis Buffer immediately before use.

Plant Tissue Plant tissue may need to be ground with a mortar and pestle in liquid nitrogen,

then homogenized with a Dounce homogenizer to improve cell breakage.

1. Harvest ~400 mg plant tissue and quickly freeze in liquid nitrogen.

2. Place tissue in a mortar precooled with liquid nitrogen. Cover the tissue with more liquid nitrogen and grind with a pestle until a fine powder is obtained. Add more liquid nitrogen as needed to keep the tissue covered with liquid nitrogen while grinding.

3. Transfer tissue and liquid nitrogen to a sterile, microcentrifuge tube. As soon as the liquid nitrogen evaporates, add 1 mL Micro-FastTrack™ 2.0 Lysis Buffer with Proteinase K (page 7).

4. Quickly homogenize the tissue using a 1-mL Dounce homogenizer until homogenous with no visible particulate matter (~10–12 strokes).

5. Proceed to the Basic mRNA Isolation Method, Step 1, page 11.

Isolating mRNA from Total RNA

The 25 mg aliquot of oligo(dT) cellulose binds ~25 μg polyA+ RNA. Assuming total RNA is 1–5% polyA+, we recommend that you start with 500 μg of total RNA per mRNA isolation.

1. Ethanol precipitate total RNA and wash with 70% ethanol.

2. Resuspend the pellet in 10 μL Elution Buffer.

3. Add the RNA solution to 1 mL Micro-FastTrack™ 2.0 Binding Buffer in a sterile, microcentrifuge tube.

4. Heat to 65°C for 5 minutes, then place immediately on ice for exactly 1 minute.

5. Proceed to Step 4 of the Basic mRNA Isolation Method, page 11.

11

Basic mRNA Isolation Method

Introduction A typical mammalian cell contains about 10-11 g of RNA of which 1–5% is polyA+

RNA. The remaining RNA is mostly rRNA (80–85% of total) and low-molecular weight RNAs such as tRNA (15–20% of total). To separate the heterogeneous population of mRNA from the majority of the RNA found in the cell, affinity binding to oligo(dT) cellulose is used. This method exploits the major characteristic of mRNA, polyadenylation, to obtain intact, pure mRNA.

The procedure described below is designed to remove DNA and proteins from your sample and allow selective binding of mRNA under high salt conditions to oligo(dT) cellulose. The resin is then transferred to a microcentrifuge spin column, and the rRNA is removed by washing the resin with a low salt buffer. The mRNA is then eluted with a very low ionic strength buffer. Intact, pure mRNA is isolated because of:

• Gentle lysis of the cells

• Efficient inactivation of RNases

• Specificity of the oligo(dT) cellulose

• Minimal handling of the mRNA sample

Note The binding capacity of oligo(dT) cellulose is ~1 μg polyA+ RNA per mg of resin.

Be sure that the amount of sample that you are adding to the oligo(dT) cellulose does not exceed its capacity. If your sample is very viscous, you may want to split the sample into 2 tubes.

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• If you are isolating polyA+ RNA from yeast cells, note that the yield of polyA+ RNA is likely to be lower than the yields obtained with other eukaryotic cells. Yeast cells generally yield lower amounts of polyA+ RNA than other eukaryotic cells because:

- Yeast mRNAs contain shorter polyA+ tails

- Yeast cells are harder to lyse

• To increase the yield of polyA+ RNA, we recommend preparing total yeast RNA using PureLink® Total RNA Mini Kit (see page 20).

• When binding to oligo(dT) cellulose, we recommend that you increase the incubation time of your RNA with oligo(dT) cellulose to at least 1 hour. Increasing the incubation time of yeast RNA with the oligo(dT) cellulose may increase the binding efficiency.

Before Starting • Equilibrate a water bath to 45°C (preferably a slow-shaking water bath)

• Obtain dry ice for mRNA precipitation

12

Basic mRNA Isolation Method, Continued

Isolating mRNA 1. Incubate the cell lysate produced in Preparing Samples, pages 7–10, at 45°C for 15-20 minutes. If insoluble material persists, centrifuge at 4000 × g for 5 minutes at room temperature and transfer the supernatant to a new sterile, microcentrifuge tube.

Note: Incubation is important for complete digestion of proteins and ribonucleases. Incubation for 20 minutes is recommended for tissues, while cell material is effectively digested with a 15 minute incubation. You may optimize the time of incubation for your particular sample.

2. Adjust NaCl concentration of the lysate to 0.5 M final concentration by adding 63 μL 5 M NaCl stock solution to each 1 mL lysate containing 0.2 M NaCl. Mix thoroughly.

3. Shear any remaining DNA by passing the lysate 3–4 times (or more as needed) through a sterile plastic 1-cc syringe fitted with an 18–21 gauge needle.

This yields a cleaner mRNA preparation. If solution is viscous, see Troubleshooting page 16.

4. Remove a vial of oligo(dT) cellulose from the dessicator and add cell lysate or total RNA solution (from Step 4, page 10) to the vial. Be sure to label the tube with the name of your sample.

5. Seal the tube and allow the oligo(dT) cellulose to swell for 2 minutes.

6. Rock the tube gently at room temperature for 15–20 minutes (using a rocking platform or a rotator). For yeast RNA, increase the rocking time to at least 1 hour (see Recommendation, page 11).

Rocking or rotating increases the efficiency of mRNA binding to oligo(dT) cellulose.

7. Centrifuge oligo(dT) cellulose at 4000 × g in a microcentrifuge for 5 minutes at room temperature. Remove the supernatant carefully from the resin bed, taking care not to disturb the oligo(dT) cellulose pellet. Proceed to Washing Oligo(dT) Cellulose, page 13.

13


Washing Oligo(dT) Cellulose

1. Resuspend the oligo(dT) cellulose in 1.3 mL Binding Buffer. Centrifuge at 4000 × g in a microcentrifuge for 5 minutes at room temperature. Remove the Binding Buffer from the resin bed.

2. Repeat Step 1 until the buffer is no longer cloudy (at least 2 more times). Use 1.3 mL Binding Buffer per wash.

3. Resuspend the resin in 0.3 mL Binding Buffer and transfer the sample to a spin-column (inside the spin-column/microcentrifuge tube set). Centrifuge at 4000 × g for 10 seconds at room temperature. Repeat this process as many times as necessary to transfer all of the oligo(dT) cellulose into the spin-column.

4. Remove spin column from the microcentrifuge tube and discard the liquid inside the tube.

5. Place the spin column back into the tube and add 500 μL Binding Buffer. Centrifuge at 4000 × g for 10 seconds at room temperature. Read the OD260 of the flow-through.

6. Repeat Step 5 (at least 3 times) until the OD260 of the flow-through is <0.05.

7. Add 200 μL Low Salt Wash Buffer and gently resuspend the resin with a sterile pipette tip. Be careful not to damage the membrane of the spin-column as you will lose the resin (and your sample). Centrifuge at 4000 × g for 10 seconds at room temperature. This low salt wash removes SDS and nonpolyadenylated RNAs such as rRNAs.

8. Repeat Step 7 with an additional 200 μL Low Salt Wash Buffer. Proceed to Eluting and Precipitating mRNA, page 14.

14


Eluting and Precipitating mRNA

1. Place the spin column into a new (sterile and RNase-free) microcentrifuge tube provided in the kit.

2. Add 100 μL Elution Buffer and mix the buffer into the cellulose bed with a sterile pipette tip.

3. Centrifuge at 4000 × g for 10 seconds at room temperature, but do not decant the liquid. The mRNA is now in the eluate.

4. Add a second 100 μL aliquot of Elution Buffer to the column, mix into the cellulose, and centrifuge again for 10 seconds.

Steps 2 through 4 elutes the mRNA into the microcentrifuge tube.

5. Remove the column from the tube. The tube now contains ~200 μL. Do Not Discard. This is your mRNA sample. Note: If the tube contains less than 200 μL, place the column back into the microcentrifuge tube and centrifuge again at 4000 × g for 1 minute at room temperature to collect any remaining eluate. Remove the column from the tube. If you wish to quantitate the RNA yield, see Determining RNA Yield on page 15.

6. Precipitate the mRNA with 10 μL of 2 mg/mL glycogen carrier (supplied), 30 μL of 2 M sodium acetate (supplied), and 600 μL of 200 proof (100%) ethanol. Place on dry ice for 10–15 minutes.

7. Centrifuge in a microcentrifuge at maximum speed for 15 minutes at 4°C. Carefully remove the supernatant and discard it without disturbing the pellet.

8. Wash the pellet with 70% ethanol. Centrifuge again to collect any residual ethanol.

9. Remove the ethanol. Centrifuge briefly and remove traces of ethanol. Air-dry the pellet for 5–10 minutes. Resuspend the mRNA pellet in 1–10 μL of Elution Buffer (10 mM Tris-HCl, pH 7.5). Use the mRNA immediately or store indefinitely at –80°C.

15


Determining RNA Yield

To determine the concentration of the eluted mRNA, dilute the sample 2-fold by adding 50 μL of sample (from Step 5, page 14) to 50 μL of Elution Buffer. Use Elution Buffer to blank the spectrophotometer at 260 nm. Place the diluted sample into a 100 μL quartz microcuvette and read the absorbance at 260 nm. Use the formula below to determine the RNA concentration:

[RNA] = (A260) (0.04 μg/μL) D

D is the dilution factor (D = 2 in the above example). Determine the RNA yield by multiplying the concentration by the volume of the RNA sample. Note that the A260 must be >0.05 to give an accurate RNA concentration.

Uses for PolyA+ RNA

The polyA+ RNA isolated from your cells or tissue of interest may be used for a variety of procedures including RT-PCR, Northern blot analysis, cDNA synthesis, RNase protection assays, oocyte microinjection, and in vitro translation. For your convenience, we have included a sample protocol for performing RT-PCR in the Appendix (see page 17).

16

Troubleshooting

Problem Reason Solution

Low yields of mRNA Proteinase K depleted because large amounts of protein in the sample compete out RNase

Reduce the amount of starting material.

mRNA is not completely eluted off oligo(dT) cellulose

Perform another set of elutions.

Heat the elution buffer to 65°C and pass over the column.

Ruptured membrane in spin column (loss of oligo(dT) cellulose)

Be careful when resuspending oligo(dT) cellulose.

mRNA is contaminated with rRNA

Poor removal of rRNA during Low Salt Wash

Mix the Wash buffers gently into the oligo(dT) cellulose with the pipette tip.

Viscous lysate Sample too large (mRNA binds to oligo(dT) cellulose if the viscosity is reduced)

Add 1 mL more of Micro-FastTrack™ 2.0 Lysis Buffer and process as 2 samples. Remember to use 2 spin-columns and 2 aliquots of oligo(dT) cellulose.

Large amounts of DNA Split the sample as above and shear with a syringe fitted with an 18–21 gauge needle.

17

Appendix

RT-PCR Protocol

Introduction A sample protocol for performing RT-PCR using the SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase is provided (page 19). The SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase is designed for the sensitive, reproducible, end-point detection and analysis of RNA molecules by RT-PCR. Using this convenient one-step formulation, you can perform both cDNA synthesis and PCR amplification in a single tube using gene-specific primers and target RNAs from either total RNA or mRNA. The system uses a mixture of SuperScript® III Reverse Transcriptase and Platinum® Taq DNA polymerase in an optimized reaction buffer, and can detect a wide range of RNA targets, from 200 bp to 4.5 kb. The amount of starting material can range from 0.01 pg to 1 μg of total RNA.

Materials Needed Wear sterile gloves for all steps. • SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA

Polymerase (page 20) • Ice bucket with ice • Thermal cycler • Autoclaved deionized water

Important Parameters and Guidelines

RNA • High quality intact RNA is essential for successful full-length cDNA

synthesis. • For low copy-number genes or longer targets, use more starting material

(>10 ng total RNA). • RNA should be devoid of any RNase contamination and aseptic conditions

should be maintained. • We recommend the PureLink® Total RNA Mini Kit or TRIzol® Reagent for

isolation of total RNA. Oligo(dT) selection for poly(A)+ RNA is typically not necessary, although it may improve the yield of specific cDNAs.

Primers • We recommend using gene specific primers (GSPs). We do not recommend

using oligo(dT) or random primers, as they can generate non-specific products in the one-step procedure and the amount of RT-PCR product may be reduced.

• A final primer concentration of 0.2 μM for each primer is generally optimal. However, for best results, we recommend performing a primer titration of 0.15–0.5 μM.

• Design primers that anneal to the mRNA sequence in exons on both sides of an intron or exon/exon boundary, to allow differentiation between the amplified cDNA and potential contaminating genomic DNA.

• Primers should not be self-complementary or complementary to each other at the 3′ ends.

18

RT-PCR Protocol, Continued

Important Parameters and Guidelines, Continued

Magnesium and dNTP concentration• MgSO4 is included in the 2X Reaction Mix at a final concentration of 1.6 mM,

which works well for most targets. If needed, the magnesium concentration can further be optimized (usually between 1.4–2 mM) with the 5-mM MgSO4 provided in the kit.

• dNTPs are included in the 2X Reaction Mix at a final concentration of 200 μM, which is optimal for most reactions.

Reactions • Program the thermal cycler before setting up the reaction. The thermal cycler

should be preheated to 45°C to 60°C, depending on the temperature selected for cDNA synthesis.

• For difficult or high GC-content templates, use a 60°C cDNA synthesis temperature.

• Keep all components, reaction mixes, and samples on ice. After preparation of the samples, transfer them to the preheated thermal cycler and immediately start the RT-PCR program.

• Efficient cDNA synthesis can be accomplished in a 15–30 min incubation at 45°C to 60°C. For small targets, an incubation time of 5 min may be sufficient.

• SuperScript™ II RT is inactivated, Platinum® Taq DNA polymerase is reactivated and the RNA/cDNA hybrid is denatured during the 2 min incubation at 94°C.

• The annealing temperature should be 10°C below the melting temperature of the primers used.

• The extension time varies with the size of the amplicon (approximately 1 min per 1 kb of amplicon).

• For all targets up to 4.5 kb, 2 μL of SuperScript™ III RT/ Platinum® Taq Mix is sufficient.

RT-PCR Protocol The following cycling conditions were established and tested using a GeneAmp®

PCR System 9600 and 2400 and a DNA Engine® PTC-200. You may need to adjust these conditions for other thermal cyclers.

Efficient cDNA synthesis can be achieved in a 15–30 minute incubation at 45°C to 60°C. We recommend a 30-min incubation at 55°C as a general starting point. The optimal temperature for reverse transcription will depend on primer and target sequences. Cycling conditions may have to be further optimized for different sequences. Three-step cycling (separate annealing and extension steps) is required.

19



1. Program the thermal cycler so that cDNA synthesis is followed immediately by PCR amplification, as follows:

cDNA synthesis 1 cycle: 45°C to 60°C for 15–30 minutes

Denaturation 1 cycle: 94°C for 2 min

PCR amplification 40 cycles: 94°C for 15 s (denature)

55°C to 65°C for 30 s (anneal) 68°C for 1 min/kb (extend)

Final extension (optional) 1 cycle: 68°C for 5 min

2. Add the following to a 0.2-mL, nuclease-free, thin-walled PCR tube on ice. For multiple reactions, you can prepare a master mix to minimize reagent loss and enable accurate pipetting.

Component Volume 2X Reaction Mix 25 μL Template RNA (0.01 pg–1 μg) x μL Sense primer (10 μM) 1 μL Anti-sense primer (10 μM) 1 μL SuperScript® III RT/ Platinum® Taq Mix* 2 μL Autoclaved distilled water to 50 μL

*You can verify absence of genomic DNA in RNA preparations by omitting the SuperScript® III RT/Platinum® Taq Mix and substituting 2 units of Platinum® Taq DNA polymerase in the reaction.

3. Gently mix and make sure that all the components are at the bottom of the amplification tube. Centrifuge briefly if needed. Depending on the thermal cycler used, overlay with silicone oil if necessary.

4. Place the reaction in the preheated thermal cycler programmed as described above. Collect the data and analyze the results.

Cloning PCR Products

Once you have completed the PCR reaction using the protocol above, you may clone your PCR products into any vector of choice. We recommend using the TOPO® TA Cloning® Kit available from Life Technologies (see page 20 for ordering information) for 5-minute cloning of your Taq-amplified PCR products into a vector suitable for screening and isolation of recombinants. Many other TOPO® TA Cloning® kits are available to allow cloning and expression of your PCR products in prokaryotes, insects, and mammalian cells. For more information, visit www.lifetechnologies.com or call Technical Support (see page 21).


20

Accessory Products

Introduction The following products are designed for use with, or in place of, Micro-FastTrack™ 2.0 kit. For details, visit www.lifetechnologies.com or contact Technical Support (page 21).

Item Amount Catalog No.

FastTrack® 2.0 Kit 6 reactions K1593-02

18 (3 × 6) reactions

K1593-03

Oligo dT Cellulose 1 g R545-01

Microcentrifuge Spin Columns with microcentrifuge tubes

50 columns R500-50

200 columns R500-20

RNase AWAY® Reagent 250 mL 10328-011

GeneRacer® Kit with SuperScript® III RT and TOPO® TA Cloning® Kit for Sequencing

1 kit L1502-01

SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase

25 reactions 12574-018

SuperScript® III First-Strand Synthesis System For RT-PCR


ThermoScript™ RT-PCR System plus Platinum® Taq DNA Polymerase


Cloned AMV First-Strand cDNA Synthesis Kit 25 reactions 12328-032

SuperScript® Choice System for cDNA Synthesis 3 reactions 18090-019

TOPO® TA Cloning® Kit 20 reactions K4500-01

PureLink® Total RNA Mini Kit 50 preps 12183-018A


21

Technical Support

Obtaining Support For the latest services and support information for all locations, go to www.lifetechnologies.com. At the website, you can:

• Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities

• Search through frequently asked questions (FAQs)

• Submit a question directly to Technical Support ([email protected])

• Search for user documents, SDSs, vector maps and sequences, application notes, formulations, handbooks, certificates of analysis, citations, and other product support documents

• Obtain information about customer training

• Download software updates and patches

Safety Data Sheets (SDS)

Safety Data Sheets (SDSs) are available at www.lifetechnologies.com/support.

Certificate of Analysis

The Certificate of Analysis provides detailed quality control and product qualification information for each product. Certificates of Analysis are available on our website. Go to www.lifetechnologies.com/support and search for the Certificate of Analysis by product lot number, which is printed on the box.

Limited Product Warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies’ General Terms and Conditions of Sale found on Life Technologies’ website at www.lifetechnologies.com/termsandconditions. If you have any questions, please contact Life Technologies at www.lifetechnologies.com/support.


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22

References

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (Ed.). (1994) Current Protocols in Molecular Biology (New York: Greene Publishing Associates and Wiley-Interscience).

Sambrook, J., Fritsch, E. F., Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).

© 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

Disclaimer LIFE 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.

26 September 2012

Headquarters5791 Van Allen Way | Carlsbad, CA 92008 USA | Phone +1 760 603 7200 | Toll Free in USA 800 955 6288For support visit lifetechnologies.com/support or email [email protected]

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