extract-n-amp™ tissue pcr kit: rapid genomic dna extraction from tissue coupled with pcr
DESCRIPTION
A feature article from Sigma-Aldrich on Extract-N-Amp™ Tissue PCR Kit: Rapid Genomic DNA Extraction from Tissue Coupled with PCR.TRANSCRIPT
Figure 1 Overview of REDExtract-N-Amptrade Tissue PCR Procedure
Materials and MethodsAll materials were obtained from Sigma-Aldrich (St Louis MO) unlessotherwise noted All PCR primers were obtained from Sigma-Genosys(The Woodlands TX)
Add Tissue Sample toTissue Preparation Solution
and Extraction Solution
Incubate for 10 minutes at room temperature
Heat 95 degC for 3 min
Add Neutralization Solution
Mix aliquot withREDExtract-N-Amptrade
PCR ReadyMixtrade and primers
Transfer to thermal cycler
PCR
Directly load amplified PCRproduct on gel
Extract-N-Amptrade TissuePCR Kit Rapid GenomicDNA Extraction fromTissue Coupled with PCRBy Scott Weber and Derek DouglasSigma-Aldrich Corporation St Louis MO USA
IntroductionStandard methods for extracting DNA from tissues can beextremely laborious and time consuming Certain appli-cations such as genotyping of transgenic mice using asection of tail employ a lengthy DNA extraction processFor example traditional mouse tail DNA extractions requirea 5 hour to overnight digestion with proteinase K andsubsequent phenolchloroform extraction and alcoholprecipitation Recently other options such as silica bindand elute kits have become available but these alsorequire a 5 hour to overnight digestion of the tail beforethe DNA is extracted These time consuming and laborintensive methods are necessary for some applicationsbut for more robust techniques such as PCR the DNAdoes not need to be isolated to the degree that thesemethods provide
PCR can amplify a target using very little template andfrom relatively impure starting material Therefore theExtract-N-Amptrade Tissue PCR Kit has been created to releasePCR-ready DNA from mouse tails in a 15 minute singletube procedure The included 2x PCR mix is optimized towork with the crude extracts and the solutions in the kit
We describe a new method from which sufficient DNAfor PCR amplification is released from a mouse tail aftera 10 min extraction The neutralized tissue extract is stablefor at least 6 months at 4 degC and can be added directlyto the optimized PCR mix which is supplied in the kitFurthermore the Extract-N-Amptrade Tissue Kit works witha variety of source materials including fresh and frozenmouse tails mouse tissue buccal swabs hair saliva andsaliva archived on a collection card
feature articles
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DNA extractionExtraction Solution and Tissue Preparation Solution weremixed together and tissue samples were added to themixture (Figure 1) The mixture was incubated at roomtemperature for 10 minutes to extract the DNA An incu-bation at 95 degC for 3 minutes stopped the extraction andNeutralization Solution B was then added to neutralize theextract Samples were mixed thoroughly and used imme-diately in PCR The volumes of the solutions used and incu-bation times for each starting material are listed in Table 1
PCR amplificationFor standard PCR 10 ml of REDExtract-N-Amptrade PCRReadyMix was combined with forward and reverse primersat 04 mM and 4 ml of neutralized tissue extract in a finalvolume of 20 ml Reactions were assembled at roomtemperature Cycling was performed in a GeneAmpreg
PCR System 9700 thermal cycler (Perkin ElmerAppliedBiosystems Foster City CA) The PCR primers used foreach sample type and the cycling parameters used arelisted in Table 1 The sequences for the primers are pre-sented in Table 2 Aliquots of 3-6 ml of each PCR productwas loaded and fractionated on an ethidium bromide-stained 1 agarose gel in Figures 2 and 3
For quantitative PCR 10 ml of Extract-N-Amptrade PCRReadyMix was combined with forward and reverse primersat 04 mM SYBRreg Green I dye (Product Code S 9430) 06 mlof Reference Dye for Quantitative PCR (Product Code R 4526) and 4 ml of tissue extract in a final volume of 20 ml Quantitative PCR was performed in an ABI PRISMreg
7700 Sequence Detection System (Perkin ElmerAppliedBiosystems Foster City CA) Primers and cycling parametersused are listed in Table 1
Mouse DNA standards for quantitative PCR were pre-pared from 12 cm of mouse tail using the GenElutetradeMammalian Genomic DNA Kit (Product Code G1N10G1N70 or G1N350) The purified DNA was diluted to 575 ngml and seven 2-fold serial dilutions were preparedfrom the initial dilution Single-use aliquots of these DNAstandard dilutions were stored at ndash20 degC A set of standardswas assayed along with the mouse tail extracts
SequencingPCR products prepared from mouse-tail extracts with theIL-1b primer pair were sequenced after purification withthe GenElutetrade PCR Clean-up Kit (Product Code NA1020)The sequence was obtained using BigDyetrade TerminatorChemistry on an ABI PRISMreg 377 DNA Sequencer(Applied Biosystems Foster City CA)
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Results and Discussion
PCR amplification from tissue extractsThe Extract-N-Amptrade Tissue PCR Kit was used to extractand amplify target DNA from mouse tails mouse braintissue buccal swabs human hair saliva and salivaarchived on a collection card As shown in Figure 2 PCRproducts ranging from 1-2 kb were detected on ethidiumbromide stained 1 agarose gel from each extract Thedata in Figure 2 demonstrates that PCR-ready DNA canbe rapidly extracted from a variety of tissue sourcesAdditionally experiments are in progress using theExtract-N-Amptrade Tissue PCR Kit to extract DNA fromother starting materials such as Drosophila and fungi
Figure 2 PCR analysis of genomic DNA extracted from various types of sourcesExtract-N-Amptrade Tissue PCR Kit was used to extract and amplify genomic DNA fromvarious sources Genomic DNA was extracted using the protocol as described inMaterials and Methods In all cases the entire extraction procedure was completed in less then 15 minutes All samples were then amplified using the specially formulatedExtract-N-Amp PCR ReadyMixtrade included in the kit PCR products generated are 1181 bp for the Interleukin-1b (IL-1b) gene in mouse and 1820 bp for the Carnitinepalmitoyltransferase II (CPT-2) in human
It is known that both the reaction conditions and thetemplate quality determine the size of PCR product thatcan be amplified1 Using the Extract-N-Amptrade Tissue PCRKit a 5 kb fragment of b-Globin (Figure 3) was successfullyamplified using extracts from buccal swab hair salivaand saliva archived on a collection card The results shownin Figures 2 and 3 demonstrate that reaction conditionsand template quality obtained with the Extract-N-Amptrade
M
2 kb mdash
15 kb mdash
1 kb mdash
750 bp mdash
500 bp mdash
300 bp mdash
MMo
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-1
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-1
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Figure 4 Quantitative PCR results showing mouse tail extract stability Four mousetails were extracted using the Extract-N-Amp Tissue PCR Kit following the proceduredescribed in Materials and Methods Plot A shows the amplification of four mouse tailextracts immediately following extraction Plot B shows the amplification of the stan-dards used to quantify the DNA in the mouse tail extracts Reaction analyses wereperformed on an ABI Prismtrade 7700reg Sequence Detection System
Half of the remaining extracts were stored at 4 degC (recom-mended storage conditions) and the other half at 37 degC(accelerated storage) Quantitative PCR was performedafter 5 and 10 weeks for all extracts and on separatealiquots of the same standards used at time zero Asshown in Figure 5 mouse-tail extracts are stable at 37 degCfor 10 weeks Results for extracts stored at 4 degC are verysimilar to those shown in Figure 5 (data not shown) Itshould be noted that the mouse tail was removed fromthe neutralized extract before storage
Figure 3 PCR analysis of genomic DNA extracted from various types of human tissueand amplified with 5 kb b-Globin primers Extract-N-Amptrade Tissue PCR Kit was usedto extract and amplify genomic DNA from various human tissue sources GenomicDNA was extracted from buccal swabs hair saliva and saliva archived on a collectioncard using the protocol as described in Materials and Methods All samples were thenamplified using the specially formulated Extract-N-Amp PCR ReadyMixtrade included in thekit The PCR products generated are 5 kb for a segment of the Human b-Globin gene
Tissue PCR Kit are suitable for amplification of both small(lt 2 kb) and medium (5 kb) sized PCR products
It should be noted that the Extract-N-Amptrade ExtractionSolution contains components that are not compatiblewith standard PCR mixes The 2x Extract-N-Amptrade PCRMixes are specifically formulated to compensate forthese components and other impurities released duringthe extraction process Extracts added to a PCR mixtureother than the Extract-N-Amptrade or REDExtract-N-AmptradePCR ReadyMix will likely yield little or no PCR product
Stability of mouse tail extractsTo test the stability of the DNA extracts prepared withthe Extract-N-Amptrade Tissue PCR Kit stability tests wereset up with mouse-tail extracts utilizing quantitative realtime PCR Four mouse tails were extracted according to theprocedure outlined in the Materials and Methods sectionand 4-ml aliquots were analyzed immediately by quantita-tive PCR with SYBRreg Green dye detection on an ABI PRISMreg
7700 The Extract-N-Amptrade Tissue PCR Kit extracts areamplified without significant quenching of the SYBR Greensignal As seen in Figure 4 the amplification plots for theextracts (A) fall in the middle of the amplification plotsfor the genomic mouse DNA standards (B) s
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feature article continued
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B1
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E1
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Figure 5 Stability of DNA in mouse tail Two mouse tails were extracted using theprocedure described in Materials and Methods Mouse tails were removed from sam-ples prior to storage The extracts were stored at 37 degC (accelerated) and quantitativePCR was repeated after 5 and 10 weeks with the extracts
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Results from stability experiments performed on the mousetail extracts using the Extract-N-Amptrade Tissue PCR Kitindicate that DNA will be detectable beyond the recom-mended storage time of 6 months at 4 degC Additionallyexperimental data suggests that the extracted DNA shouldbe detectable up to 12 months after the original extraction
Sequencing of PCR products from mouse tail extractsPCR products produced with the Extract-N-Amptrade TissuePCR Kit can be sequenced directly however higher qualitysequence is often obtained after purifying the productthrough a routine clean-up procedure As illustrated inFigure 6 a clean trace was obtained from mouse-tail extractsby sequencing with the original primers (IL-1b) used forPCR Prior to sequencing the PCR product was cleanedup using the GenElutetrade PCR Clean-Up Kit (Product CodeNA1020) The nucleotide sequence was checked againstthe published sequence (NCBI Accession Number X04964)and found to be accurate
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50
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NA
Extract
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Figure 6 Sequence determination for 1181 bp Interleukin-1b (IL-1b) mouse tail PCR product PCR product was purified with the GenElutetrade PCR Clean-Up Kit The DNAextraction and PCR were performed using Sigmarsquos Extract-N-Amptrade Tissue PCR Kit The sequence was obtained using ABI BigDyetrade Terminator Chemistry and the sameprimers as for the original PCR Reaction products were resolved on an ABI PRISMreg 377 DNA Sequencer
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SummaryThe Extract-N-Amptrade Tissue PCR Kit extracts DNA suitablefor PCR in 15 minutes using a straightforward protocolWhile most commonly used with mouse tails in genotypingexperiments the kit has been shown to work with a vari-ety of starting materials including mouse tissues buccalswabs hair saliva saliva archived on a collection cardand C elegans DNA prepared with the Extract-N-AmptradeTissue PCR Kit is stable for at least 6 months at 4 degC allowingfor multiple reassays Short to medium-sized PCR targets(up to 5 kb) can be amplified from the extracts on a con-sistent basis Furthermore the PCR products are suitablefor sequencing
AcknowledgementsThe authors thank Jessica Copeland Jaime Miller andBruce Blackerby of Sigma-Aldrich Biotechnology RampD forproviding the sequence data Kevin Ray and Carol Kreaderfor their critical reading and contributions to the prepa-ration of this article Also thanks to Dave Hanson andSudhir Nayak from the Tim Schedl lab at WashingtonUniversity St Louis MO
References1 Dieffenbach Carl W and Dveksler Gabriela S (eds) PCR Primer A
Laboratory Manual p 3 (Cold Spring Harbor Laboratory Press 1995)2 Smial D et al Rapid cost-effective gene mutation screening for carni-
tine palmitoyltransferase II deficiency using whole blood on filterpaper Clinical Chemistry 45 2035-2038 (1999)
3 Lin Z and Floros J Protocol for genomic DNA preparation from freshor frozen serum for PCR amplification BioTechniques 29 460-466(2000)
Refer to Table 1 and Table 2 on next page for PCR primers used for eachsample type cycling parameters and sequences for the primers
Ordering InformationExtractions
Product Description Amplifications
XNATS REDExtract-N-Amp Tissue PCR Kit 1010
XNAT REDExtract-N-Amp Tissue PCR Kit 100100
XNATR REDExtract-N-Amp Tissue PCR Kit 10001000
XNAT2 Extract-N-Amp Tissue PCR Kit 100100
XNAT2R Extract-N-Amp Tissue PCR Kit 10001000
E 3004- Extract-N-Amp Tissue PCR mdash12 ml Reaction Mix 1000
R 4775- REDExtract-N-Amp Tissue PCR mdash12 ml Reaction Mix 1000
feature article continued
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Table 1 DNA Extraction and PCR Conditions
Table 2 Primer Size and Sequence Information
Extraction Reference to Quantitative Tissue Type Volumes Primers PCR Conditions Agarose Gel PCR ConditionsMouse Tail 100 ml Ext Il-1b 94 degC 3 min (Figure 2) 94 degC 3 min
25 ml Tissue Prep 35 cycles of 40 cycles of 05-1 cm of 100 ml Neut B 94 degC 45 sec 94 degC 30 secmouse tail tip 58 degC 1 min 58 degC 30 sec
72 degC 2 min 72 degC 15 minMouse Brain Tissue followed by (Figure 2) mdash
72 degC 7 min5 mg frozen mouse brainBuccal Swab 200 ml Ext CPT21 and CPT2 and SPB CPT2 94 degC 3 min
25 ml Tissue Prep b-Globin 94 degC 3 min (Figure 2) 40 cycles of 200 ml Neut B for standard 35 cycles of 94 degC 30 sec
PCR SPB2 for 94 degC 30 sec b-Globin 60 degC 1 minQuantitative 60 degC 1 min (Figure 3) 72 degC 2 minPCR 72 degC 2 min
Hair 100 ml Ext CPT21 and followed by CPT2 mdash25 ml Tissue Prep b-Globin 72 degC 7 min (Figure 2)
1 hair shaft root end 100 ml Neut Bin solution b-Globin 5 kb b-Globin
94 degC 5 sec (Figure 3)Saliva 45 cycles of CPT2 mdash
94 degC 5 sec (Figure 2) 10 ml of fresh saliva 68 degC 7 min
followed by b-Globin72 degC 7 min (Figure 3)
Saliva archived on a CPT2 mdashcollection card (Figure 2)
18rdquo punch of dried b-Globinsaliva on card (Figure 3)C elegans 100 ml Ext rrf-3 94 degC 3 min (Figure 2) mdash
25 ml Tissue Prep 35 cycles of 1 C elegans in solution 100 ml Neut B 94 degC 30 sec
(Extraction 58 degC 1 minincubated at 72 degC 15 min55 degC for followed by 10 minutes) 72 degC 7 min
Primer Name Size Forward Sequence Reverse SequenceIL-1b(Interleukin 1 Beta)1 1181 bp 5rsquo-TCTGGGGTTGATGTAGGA-3rsquo 5rsquo-GGGCTGGAAAAATGGTC-3rsquob-Globin2 5000 bp 5rsquo-CCTCAGCCTCAGAATTTGGCA-3rsquo 5rsquo-TCTCCCCAACCTCCCCCATCTA-3rsquoCPT21 1820 bp 5rsquo-CTGTCAGCCTTACACTGACCC-3rsquo 5rsquo-AGGAAGGGAGGATGAGACGT-3rsquo(carnitine palymitoyltransferase II)
SPB2 547 bp 5rsquo-CTCGAATCCCCAGCACCCTTCATTTC 5rsquo-GAGGTGCCATGGCTGAGTCA-3rsquo(human surfactant protein B) AGA-3rsquorrf-33 1079 bp 5rsquo-CACTCTTCATCGTCGGCTTTTC-3rsquo 5rsquo-CATCGCGAAGAGTCAATGTTTG-3rsquo
1 NCBI Accession Number X049642 NCBI Accession Number U013173 Wormbase gene designation F10B57
7
3
DNA extractionExtraction Solution and Tissue Preparation Solution weremixed together and tissue samples were added to themixture (Figure 1) The mixture was incubated at roomtemperature for 10 minutes to extract the DNA An incu-bation at 95 degC for 3 minutes stopped the extraction andNeutralization Solution B was then added to neutralize theextract Samples were mixed thoroughly and used imme-diately in PCR The volumes of the solutions used and incu-bation times for each starting material are listed in Table 1
PCR amplificationFor standard PCR 10 ml of REDExtract-N-Amptrade PCRReadyMix was combined with forward and reverse primersat 04 mM and 4 ml of neutralized tissue extract in a finalvolume of 20 ml Reactions were assembled at roomtemperature Cycling was performed in a GeneAmpreg
PCR System 9700 thermal cycler (Perkin ElmerAppliedBiosystems Foster City CA) The PCR primers used foreach sample type and the cycling parameters used arelisted in Table 1 The sequences for the primers are pre-sented in Table 2 Aliquots of 3-6 ml of each PCR productwas loaded and fractionated on an ethidium bromide-stained 1 agarose gel in Figures 2 and 3
For quantitative PCR 10 ml of Extract-N-Amptrade PCRReadyMix was combined with forward and reverse primersat 04 mM SYBRreg Green I dye (Product Code S 9430) 06 mlof Reference Dye for Quantitative PCR (Product Code R 4526) and 4 ml of tissue extract in a final volume of 20 ml Quantitative PCR was performed in an ABI PRISMreg
7700 Sequence Detection System (Perkin ElmerAppliedBiosystems Foster City CA) Primers and cycling parametersused are listed in Table 1
Mouse DNA standards for quantitative PCR were pre-pared from 12 cm of mouse tail using the GenElutetradeMammalian Genomic DNA Kit (Product Code G1N10G1N70 or G1N350) The purified DNA was diluted to 575 ngml and seven 2-fold serial dilutions were preparedfrom the initial dilution Single-use aliquots of these DNAstandard dilutions were stored at ndash20 degC A set of standardswas assayed along with the mouse tail extracts
SequencingPCR products prepared from mouse-tail extracts with theIL-1b primer pair were sequenced after purification withthe GenElutetrade PCR Clean-up Kit (Product Code NA1020)The sequence was obtained using BigDyetrade TerminatorChemistry on an ABI PRISMreg 377 DNA Sequencer(Applied Biosystems Foster City CA)
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Results and Discussion
PCR amplification from tissue extractsThe Extract-N-Amptrade Tissue PCR Kit was used to extractand amplify target DNA from mouse tails mouse braintissue buccal swabs human hair saliva and salivaarchived on a collection card As shown in Figure 2 PCRproducts ranging from 1-2 kb were detected on ethidiumbromide stained 1 agarose gel from each extract Thedata in Figure 2 demonstrates that PCR-ready DNA canbe rapidly extracted from a variety of tissue sourcesAdditionally experiments are in progress using theExtract-N-Amptrade Tissue PCR Kit to extract DNA fromother starting materials such as Drosophila and fungi
Figure 2 PCR analysis of genomic DNA extracted from various types of sourcesExtract-N-Amptrade Tissue PCR Kit was used to extract and amplify genomic DNA fromvarious sources Genomic DNA was extracted using the protocol as described inMaterials and Methods In all cases the entire extraction procedure was completed in less then 15 minutes All samples were then amplified using the specially formulatedExtract-N-Amp PCR ReadyMixtrade included in the kit PCR products generated are 1181 bp for the Interleukin-1b (IL-1b) gene in mouse and 1820 bp for the Carnitinepalmitoyltransferase II (CPT-2) in human
It is known that both the reaction conditions and thetemplate quality determine the size of PCR product thatcan be amplified1 Using the Extract-N-Amptrade Tissue PCRKit a 5 kb fragment of b-Globin (Figure 3) was successfullyamplified using extracts from buccal swab hair salivaand saliva archived on a collection card The results shownin Figures 2 and 3 demonstrate that reaction conditionsand template quality obtained with the Extract-N-Amptrade
M
2 kb mdash
15 kb mdash
1 kb mdash
750 bp mdash
500 bp mdash
300 bp mdash
MMo
use
Tai
l IL
-1
Mo
use
Bra
in T
issu
e IL
-1
Bu
ccal
Sw
ab C
PT-2
Hai
r C
PT-2
Saliv
a C
PT-2
Saliv
a C
ard
CPT
-2
Neg
ativ
e C
on
tro
l
A
B
Figure 4 Quantitative PCR results showing mouse tail extract stability Four mousetails were extracted using the Extract-N-Amp Tissue PCR Kit following the proceduredescribed in Materials and Methods Plot A shows the amplification of four mouse tailextracts immediately following extraction Plot B shows the amplification of the stan-dards used to quantify the DNA in the mouse tail extracts Reaction analyses wereperformed on an ABI Prismtrade 7700reg Sequence Detection System
Half of the remaining extracts were stored at 4 degC (recom-mended storage conditions) and the other half at 37 degC(accelerated storage) Quantitative PCR was performedafter 5 and 10 weeks for all extracts and on separatealiquots of the same standards used at time zero Asshown in Figure 5 mouse-tail extracts are stable at 37 degCfor 10 weeks Results for extracts stored at 4 degC are verysimilar to those shown in Figure 5 (data not shown) Itshould be noted that the mouse tail was removed fromthe neutralized extract before storage
Figure 3 PCR analysis of genomic DNA extracted from various types of human tissueand amplified with 5 kb b-Globin primers Extract-N-Amptrade Tissue PCR Kit was usedto extract and amplify genomic DNA from various human tissue sources GenomicDNA was extracted from buccal swabs hair saliva and saliva archived on a collectioncard using the protocol as described in Materials and Methods All samples were thenamplified using the specially formulated Extract-N-Amp PCR ReadyMixtrade included in thekit The PCR products generated are 5 kb for a segment of the Human b-Globin gene
Tissue PCR Kit are suitable for amplification of both small(lt 2 kb) and medium (5 kb) sized PCR products
It should be noted that the Extract-N-Amptrade ExtractionSolution contains components that are not compatiblewith standard PCR mixes The 2x Extract-N-Amptrade PCRMixes are specifically formulated to compensate forthese components and other impurities released duringthe extraction process Extracts added to a PCR mixtureother than the Extract-N-Amptrade or REDExtract-N-AmptradePCR ReadyMix will likely yield little or no PCR product
Stability of mouse tail extractsTo test the stability of the DNA extracts prepared withthe Extract-N-Amptrade Tissue PCR Kit stability tests wereset up with mouse-tail extracts utilizing quantitative realtime PCR Four mouse tails were extracted according to theprocedure outlined in the Materials and Methods sectionand 4-ml aliquots were analyzed immediately by quantita-tive PCR with SYBRreg Green dye detection on an ABI PRISMreg
7700 The Extract-N-Amptrade Tissue PCR Kit extracts areamplified without significant quenching of the SYBR Greensignal As seen in Figure 4 the amplification plots for theextracts (A) fall in the middle of the amplification plotsfor the genomic mouse DNA standards (B) s
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feature article continued
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
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E9
F9
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10^ 0
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E1
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Figure 5 Stability of DNA in mouse tail Two mouse tails were extracted using theprocedure described in Materials and Methods Mouse tails were removed from sam-ples prior to storage The extracts were stored at 37 degC (accelerated) and quantitativePCR was repeated after 5 and 10 weeks with the extracts
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Results from stability experiments performed on the mousetail extracts using the Extract-N-Amptrade Tissue PCR Kitindicate that DNA will be detectable beyond the recom-mended storage time of 6 months at 4 degC Additionallyexperimental data suggests that the extracted DNA shouldbe detectable up to 12 months after the original extraction
Sequencing of PCR products from mouse tail extractsPCR products produced with the Extract-N-Amptrade TissuePCR Kit can be sequenced directly however higher qualitysequence is often obtained after purifying the productthrough a routine clean-up procedure As illustrated inFigure 6 a clean trace was obtained from mouse-tail extractsby sequencing with the original primers (IL-1b) used forPCR Prior to sequencing the PCR product was cleanedup using the GenElutetrade PCR Clean-Up Kit (Product CodeNA1020) The nucleotide sequence was checked againstthe published sequence (NCBI Accession Number X04964)and found to be accurate
30
25
20
15
10
5
35
40
45
0
50
ng D
NA
Extract
10 wk 37 ˚C5 wk 37 ˚Ct=0
1 2
Figure 6 Sequence determination for 1181 bp Interleukin-1b (IL-1b) mouse tail PCR product PCR product was purified with the GenElutetrade PCR Clean-Up Kit The DNAextraction and PCR were performed using Sigmarsquos Extract-N-Amptrade Tissue PCR Kit The sequence was obtained using ABI BigDyetrade Terminator Chemistry and the sameprimers as for the original PCR Reaction products were resolved on an ABI PRISMreg 377 DNA Sequencer
si
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SummaryThe Extract-N-Amptrade Tissue PCR Kit extracts DNA suitablefor PCR in 15 minutes using a straightforward protocolWhile most commonly used with mouse tails in genotypingexperiments the kit has been shown to work with a vari-ety of starting materials including mouse tissues buccalswabs hair saliva saliva archived on a collection cardand C elegans DNA prepared with the Extract-N-AmptradeTissue PCR Kit is stable for at least 6 months at 4 degC allowingfor multiple reassays Short to medium-sized PCR targets(up to 5 kb) can be amplified from the extracts on a con-sistent basis Furthermore the PCR products are suitablefor sequencing
AcknowledgementsThe authors thank Jessica Copeland Jaime Miller andBruce Blackerby of Sigma-Aldrich Biotechnology RampD forproviding the sequence data Kevin Ray and Carol Kreaderfor their critical reading and contributions to the prepa-ration of this article Also thanks to Dave Hanson andSudhir Nayak from the Tim Schedl lab at WashingtonUniversity St Louis MO
References1 Dieffenbach Carl W and Dveksler Gabriela S (eds) PCR Primer A
Laboratory Manual p 3 (Cold Spring Harbor Laboratory Press 1995)2 Smial D et al Rapid cost-effective gene mutation screening for carni-
tine palmitoyltransferase II deficiency using whole blood on filterpaper Clinical Chemistry 45 2035-2038 (1999)
3 Lin Z and Floros J Protocol for genomic DNA preparation from freshor frozen serum for PCR amplification BioTechniques 29 460-466(2000)
Refer to Table 1 and Table 2 on next page for PCR primers used for eachsample type cycling parameters and sequences for the primers
Ordering InformationExtractions
Product Description Amplifications
XNATS REDExtract-N-Amp Tissue PCR Kit 1010
XNAT REDExtract-N-Amp Tissue PCR Kit 100100
XNATR REDExtract-N-Amp Tissue PCR Kit 10001000
XNAT2 Extract-N-Amp Tissue PCR Kit 100100
XNAT2R Extract-N-Amp Tissue PCR Kit 10001000
E 3004- Extract-N-Amp Tissue PCR mdash12 ml Reaction Mix 1000
R 4775- REDExtract-N-Amp Tissue PCR mdash12 ml Reaction Mix 1000
feature article continued
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Table 1 DNA Extraction and PCR Conditions
Table 2 Primer Size and Sequence Information
Extraction Reference to Quantitative Tissue Type Volumes Primers PCR Conditions Agarose Gel PCR ConditionsMouse Tail 100 ml Ext Il-1b 94 degC 3 min (Figure 2) 94 degC 3 min
25 ml Tissue Prep 35 cycles of 40 cycles of 05-1 cm of 100 ml Neut B 94 degC 45 sec 94 degC 30 secmouse tail tip 58 degC 1 min 58 degC 30 sec
72 degC 2 min 72 degC 15 minMouse Brain Tissue followed by (Figure 2) mdash
72 degC 7 min5 mg frozen mouse brainBuccal Swab 200 ml Ext CPT21 and CPT2 and SPB CPT2 94 degC 3 min
25 ml Tissue Prep b-Globin 94 degC 3 min (Figure 2) 40 cycles of 200 ml Neut B for standard 35 cycles of 94 degC 30 sec
PCR SPB2 for 94 degC 30 sec b-Globin 60 degC 1 minQuantitative 60 degC 1 min (Figure 3) 72 degC 2 minPCR 72 degC 2 min
Hair 100 ml Ext CPT21 and followed by CPT2 mdash25 ml Tissue Prep b-Globin 72 degC 7 min (Figure 2)
1 hair shaft root end 100 ml Neut Bin solution b-Globin 5 kb b-Globin
94 degC 5 sec (Figure 3)Saliva 45 cycles of CPT2 mdash
94 degC 5 sec (Figure 2) 10 ml of fresh saliva 68 degC 7 min
followed by b-Globin72 degC 7 min (Figure 3)
Saliva archived on a CPT2 mdashcollection card (Figure 2)
18rdquo punch of dried b-Globinsaliva on card (Figure 3)C elegans 100 ml Ext rrf-3 94 degC 3 min (Figure 2) mdash
25 ml Tissue Prep 35 cycles of 1 C elegans in solution 100 ml Neut B 94 degC 30 sec
(Extraction 58 degC 1 minincubated at 72 degC 15 min55 degC for followed by 10 minutes) 72 degC 7 min
Primer Name Size Forward Sequence Reverse SequenceIL-1b(Interleukin 1 Beta)1 1181 bp 5rsquo-TCTGGGGTTGATGTAGGA-3rsquo 5rsquo-GGGCTGGAAAAATGGTC-3rsquob-Globin2 5000 bp 5rsquo-CCTCAGCCTCAGAATTTGGCA-3rsquo 5rsquo-TCTCCCCAACCTCCCCCATCTA-3rsquoCPT21 1820 bp 5rsquo-CTGTCAGCCTTACACTGACCC-3rsquo 5rsquo-AGGAAGGGAGGATGAGACGT-3rsquo(carnitine palymitoyltransferase II)
SPB2 547 bp 5rsquo-CTCGAATCCCCAGCACCCTTCATTTC 5rsquo-GAGGTGCCATGGCTGAGTCA-3rsquo(human surfactant protein B) AGA-3rsquorrf-33 1079 bp 5rsquo-CACTCTTCATCGTCGGCTTTTC-3rsquo 5rsquo-CATCGCGAAGAGTCAATGTTTG-3rsquo
1 NCBI Accession Number X049642 NCBI Accession Number U013173 Wormbase gene designation F10B57
7
A
B
Figure 4 Quantitative PCR results showing mouse tail extract stability Four mousetails were extracted using the Extract-N-Amp Tissue PCR Kit following the proceduredescribed in Materials and Methods Plot A shows the amplification of four mouse tailextracts immediately following extraction Plot B shows the amplification of the stan-dards used to quantify the DNA in the mouse tail extracts Reaction analyses wereperformed on an ABI Prismtrade 7700reg Sequence Detection System
Half of the remaining extracts were stored at 4 degC (recom-mended storage conditions) and the other half at 37 degC(accelerated storage) Quantitative PCR was performedafter 5 and 10 weeks for all extracts and on separatealiquots of the same standards used at time zero Asshown in Figure 5 mouse-tail extracts are stable at 37 degCfor 10 weeks Results for extracts stored at 4 degC are verysimilar to those shown in Figure 5 (data not shown) Itshould be noted that the mouse tail was removed fromthe neutralized extract before storage
Figure 3 PCR analysis of genomic DNA extracted from various types of human tissueand amplified with 5 kb b-Globin primers Extract-N-Amptrade Tissue PCR Kit was usedto extract and amplify genomic DNA from various human tissue sources GenomicDNA was extracted from buccal swabs hair saliva and saliva archived on a collectioncard using the protocol as described in Materials and Methods All samples were thenamplified using the specially formulated Extract-N-Amp PCR ReadyMixtrade included in thekit The PCR products generated are 5 kb for a segment of the Human b-Globin gene
Tissue PCR Kit are suitable for amplification of both small(lt 2 kb) and medium (5 kb) sized PCR products
It should be noted that the Extract-N-Amptrade ExtractionSolution contains components that are not compatiblewith standard PCR mixes The 2x Extract-N-Amptrade PCRMixes are specifically formulated to compensate forthese components and other impurities released duringthe extraction process Extracts added to a PCR mixtureother than the Extract-N-Amptrade or REDExtract-N-AmptradePCR ReadyMix will likely yield little or no PCR product
Stability of mouse tail extractsTo test the stability of the DNA extracts prepared withthe Extract-N-Amptrade Tissue PCR Kit stability tests wereset up with mouse-tail extracts utilizing quantitative realtime PCR Four mouse tails were extracted according to theprocedure outlined in the Materials and Methods sectionand 4-ml aliquots were analyzed immediately by quantita-tive PCR with SYBRreg Green dye detection on an ABI PRISMreg
7700 The Extract-N-Amptrade Tissue PCR Kit extracts areamplified without significant quenching of the SYBR Greensignal As seen in Figure 4 the amplification plots for theextracts (A) fall in the middle of the amplification plotsfor the genomic mouse DNA standards (B) s
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0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
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Figure 5 Stability of DNA in mouse tail Two mouse tails were extracted using theprocedure described in Materials and Methods Mouse tails were removed from sam-ples prior to storage The extracts were stored at 37 degC (accelerated) and quantitativePCR was repeated after 5 and 10 weeks with the extracts
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Results from stability experiments performed on the mousetail extracts using the Extract-N-Amptrade Tissue PCR Kitindicate that DNA will be detectable beyond the recom-mended storage time of 6 months at 4 degC Additionallyexperimental data suggests that the extracted DNA shouldbe detectable up to 12 months after the original extraction
Sequencing of PCR products from mouse tail extractsPCR products produced with the Extract-N-Amptrade TissuePCR Kit can be sequenced directly however higher qualitysequence is often obtained after purifying the productthrough a routine clean-up procedure As illustrated inFigure 6 a clean trace was obtained from mouse-tail extractsby sequencing with the original primers (IL-1b) used forPCR Prior to sequencing the PCR product was cleanedup using the GenElutetrade PCR Clean-Up Kit (Product CodeNA1020) The nucleotide sequence was checked againstthe published sequence (NCBI Accession Number X04964)and found to be accurate
30
25
20
15
10
5
35
40
45
0
50
ng D
NA
Extract
10 wk 37 ˚C5 wk 37 ˚Ct=0
1 2
Figure 6 Sequence determination for 1181 bp Interleukin-1b (IL-1b) mouse tail PCR product PCR product was purified with the GenElutetrade PCR Clean-Up Kit The DNAextraction and PCR were performed using Sigmarsquos Extract-N-Amptrade Tissue PCR Kit The sequence was obtained using ABI BigDyetrade Terminator Chemistry and the sameprimers as for the original PCR Reaction products were resolved on an ABI PRISMreg 377 DNA Sequencer
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SummaryThe Extract-N-Amptrade Tissue PCR Kit extracts DNA suitablefor PCR in 15 minutes using a straightforward protocolWhile most commonly used with mouse tails in genotypingexperiments the kit has been shown to work with a vari-ety of starting materials including mouse tissues buccalswabs hair saliva saliva archived on a collection cardand C elegans DNA prepared with the Extract-N-AmptradeTissue PCR Kit is stable for at least 6 months at 4 degC allowingfor multiple reassays Short to medium-sized PCR targets(up to 5 kb) can be amplified from the extracts on a con-sistent basis Furthermore the PCR products are suitablefor sequencing
AcknowledgementsThe authors thank Jessica Copeland Jaime Miller andBruce Blackerby of Sigma-Aldrich Biotechnology RampD forproviding the sequence data Kevin Ray and Carol Kreaderfor their critical reading and contributions to the prepa-ration of this article Also thanks to Dave Hanson andSudhir Nayak from the Tim Schedl lab at WashingtonUniversity St Louis MO
References1 Dieffenbach Carl W and Dveksler Gabriela S (eds) PCR Primer A
Laboratory Manual p 3 (Cold Spring Harbor Laboratory Press 1995)2 Smial D et al Rapid cost-effective gene mutation screening for carni-
tine palmitoyltransferase II deficiency using whole blood on filterpaper Clinical Chemistry 45 2035-2038 (1999)
3 Lin Z and Floros J Protocol for genomic DNA preparation from freshor frozen serum for PCR amplification BioTechniques 29 460-466(2000)
Refer to Table 1 and Table 2 on next page for PCR primers used for eachsample type cycling parameters and sequences for the primers
Ordering InformationExtractions
Product Description Amplifications
XNATS REDExtract-N-Amp Tissue PCR Kit 1010
XNAT REDExtract-N-Amp Tissue PCR Kit 100100
XNATR REDExtract-N-Amp Tissue PCR Kit 10001000
XNAT2 Extract-N-Amp Tissue PCR Kit 100100
XNAT2R Extract-N-Amp Tissue PCR Kit 10001000
E 3004- Extract-N-Amp Tissue PCR mdash12 ml Reaction Mix 1000
R 4775- REDExtract-N-Amp Tissue PCR mdash12 ml Reaction Mix 1000
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Table 1 DNA Extraction and PCR Conditions
Table 2 Primer Size and Sequence Information
Extraction Reference to Quantitative Tissue Type Volumes Primers PCR Conditions Agarose Gel PCR ConditionsMouse Tail 100 ml Ext Il-1b 94 degC 3 min (Figure 2) 94 degC 3 min
25 ml Tissue Prep 35 cycles of 40 cycles of 05-1 cm of 100 ml Neut B 94 degC 45 sec 94 degC 30 secmouse tail tip 58 degC 1 min 58 degC 30 sec
72 degC 2 min 72 degC 15 minMouse Brain Tissue followed by (Figure 2) mdash
72 degC 7 min5 mg frozen mouse brainBuccal Swab 200 ml Ext CPT21 and CPT2 and SPB CPT2 94 degC 3 min
25 ml Tissue Prep b-Globin 94 degC 3 min (Figure 2) 40 cycles of 200 ml Neut B for standard 35 cycles of 94 degC 30 sec
PCR SPB2 for 94 degC 30 sec b-Globin 60 degC 1 minQuantitative 60 degC 1 min (Figure 3) 72 degC 2 minPCR 72 degC 2 min
Hair 100 ml Ext CPT21 and followed by CPT2 mdash25 ml Tissue Prep b-Globin 72 degC 7 min (Figure 2)
1 hair shaft root end 100 ml Neut Bin solution b-Globin 5 kb b-Globin
94 degC 5 sec (Figure 3)Saliva 45 cycles of CPT2 mdash
94 degC 5 sec (Figure 2) 10 ml of fresh saliva 68 degC 7 min
followed by b-Globin72 degC 7 min (Figure 3)
Saliva archived on a CPT2 mdashcollection card (Figure 2)
18rdquo punch of dried b-Globinsaliva on card (Figure 3)C elegans 100 ml Ext rrf-3 94 degC 3 min (Figure 2) mdash
25 ml Tissue Prep 35 cycles of 1 C elegans in solution 100 ml Neut B 94 degC 30 sec
(Extraction 58 degC 1 minincubated at 72 degC 15 min55 degC for followed by 10 minutes) 72 degC 7 min
Primer Name Size Forward Sequence Reverse SequenceIL-1b(Interleukin 1 Beta)1 1181 bp 5rsquo-TCTGGGGTTGATGTAGGA-3rsquo 5rsquo-GGGCTGGAAAAATGGTC-3rsquob-Globin2 5000 bp 5rsquo-CCTCAGCCTCAGAATTTGGCA-3rsquo 5rsquo-TCTCCCCAACCTCCCCCATCTA-3rsquoCPT21 1820 bp 5rsquo-CTGTCAGCCTTACACTGACCC-3rsquo 5rsquo-AGGAAGGGAGGATGAGACGT-3rsquo(carnitine palymitoyltransferase II)
SPB2 547 bp 5rsquo-CTCGAATCCCCAGCACCCTTCATTTC 5rsquo-GAGGTGCCATGGCTGAGTCA-3rsquo(human surfactant protein B) AGA-3rsquorrf-33 1079 bp 5rsquo-CACTCTTCATCGTCGGCTTTTC-3rsquo 5rsquo-CATCGCGAAGAGTCAATGTTTG-3rsquo
1 NCBI Accession Number X049642 NCBI Accession Number U013173 Wormbase gene designation F10B57
7
5
Figure 5 Stability of DNA in mouse tail Two mouse tails were extracted using theprocedure described in Materials and Methods Mouse tails were removed from sam-ples prior to storage The extracts were stored at 37 degC (accelerated) and quantitativePCR was repeated after 5 and 10 weeks with the extracts
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Results from stability experiments performed on the mousetail extracts using the Extract-N-Amptrade Tissue PCR Kitindicate that DNA will be detectable beyond the recom-mended storage time of 6 months at 4 degC Additionallyexperimental data suggests that the extracted DNA shouldbe detectable up to 12 months after the original extraction
Sequencing of PCR products from mouse tail extractsPCR products produced with the Extract-N-Amptrade TissuePCR Kit can be sequenced directly however higher qualitysequence is often obtained after purifying the productthrough a routine clean-up procedure As illustrated inFigure 6 a clean trace was obtained from mouse-tail extractsby sequencing with the original primers (IL-1b) used forPCR Prior to sequencing the PCR product was cleanedup using the GenElutetrade PCR Clean-Up Kit (Product CodeNA1020) The nucleotide sequence was checked againstthe published sequence (NCBI Accession Number X04964)and found to be accurate
30
25
20
15
10
5
35
40
45
0
50
ng D
NA
Extract
10 wk 37 ˚C5 wk 37 ˚Ct=0
1 2
Figure 6 Sequence determination for 1181 bp Interleukin-1b (IL-1b) mouse tail PCR product PCR product was purified with the GenElutetrade PCR Clean-Up Kit The DNAextraction and PCR were performed using Sigmarsquos Extract-N-Amptrade Tissue PCR Kit The sequence was obtained using ABI BigDyetrade Terminator Chemistry and the sameprimers as for the original PCR Reaction products were resolved on an ABI PRISMreg 377 DNA Sequencer
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SummaryThe Extract-N-Amptrade Tissue PCR Kit extracts DNA suitablefor PCR in 15 minutes using a straightforward protocolWhile most commonly used with mouse tails in genotypingexperiments the kit has been shown to work with a vari-ety of starting materials including mouse tissues buccalswabs hair saliva saliva archived on a collection cardand C elegans DNA prepared with the Extract-N-AmptradeTissue PCR Kit is stable for at least 6 months at 4 degC allowingfor multiple reassays Short to medium-sized PCR targets(up to 5 kb) can be amplified from the extracts on a con-sistent basis Furthermore the PCR products are suitablefor sequencing
AcknowledgementsThe authors thank Jessica Copeland Jaime Miller andBruce Blackerby of Sigma-Aldrich Biotechnology RampD forproviding the sequence data Kevin Ray and Carol Kreaderfor their critical reading and contributions to the prepa-ration of this article Also thanks to Dave Hanson andSudhir Nayak from the Tim Schedl lab at WashingtonUniversity St Louis MO
References1 Dieffenbach Carl W and Dveksler Gabriela S (eds) PCR Primer A
Laboratory Manual p 3 (Cold Spring Harbor Laboratory Press 1995)2 Smial D et al Rapid cost-effective gene mutation screening for carni-
tine palmitoyltransferase II deficiency using whole blood on filterpaper Clinical Chemistry 45 2035-2038 (1999)
3 Lin Z and Floros J Protocol for genomic DNA preparation from freshor frozen serum for PCR amplification BioTechniques 29 460-466(2000)
Refer to Table 1 and Table 2 on next page for PCR primers used for eachsample type cycling parameters and sequences for the primers
Ordering InformationExtractions
Product Description Amplifications
XNATS REDExtract-N-Amp Tissue PCR Kit 1010
XNAT REDExtract-N-Amp Tissue PCR Kit 100100
XNATR REDExtract-N-Amp Tissue PCR Kit 10001000
XNAT2 Extract-N-Amp Tissue PCR Kit 100100
XNAT2R Extract-N-Amp Tissue PCR Kit 10001000
E 3004- Extract-N-Amp Tissue PCR mdash12 ml Reaction Mix 1000
R 4775- REDExtract-N-Amp Tissue PCR mdash12 ml Reaction Mix 1000
feature article continued
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Table 1 DNA Extraction and PCR Conditions
Table 2 Primer Size and Sequence Information
Extraction Reference to Quantitative Tissue Type Volumes Primers PCR Conditions Agarose Gel PCR ConditionsMouse Tail 100 ml Ext Il-1b 94 degC 3 min (Figure 2) 94 degC 3 min
25 ml Tissue Prep 35 cycles of 40 cycles of 05-1 cm of 100 ml Neut B 94 degC 45 sec 94 degC 30 secmouse tail tip 58 degC 1 min 58 degC 30 sec
72 degC 2 min 72 degC 15 minMouse Brain Tissue followed by (Figure 2) mdash
72 degC 7 min5 mg frozen mouse brainBuccal Swab 200 ml Ext CPT21 and CPT2 and SPB CPT2 94 degC 3 min
25 ml Tissue Prep b-Globin 94 degC 3 min (Figure 2) 40 cycles of 200 ml Neut B for standard 35 cycles of 94 degC 30 sec
PCR SPB2 for 94 degC 30 sec b-Globin 60 degC 1 minQuantitative 60 degC 1 min (Figure 3) 72 degC 2 minPCR 72 degC 2 min
Hair 100 ml Ext CPT21 and followed by CPT2 mdash25 ml Tissue Prep b-Globin 72 degC 7 min (Figure 2)
1 hair shaft root end 100 ml Neut Bin solution b-Globin 5 kb b-Globin
94 degC 5 sec (Figure 3)Saliva 45 cycles of CPT2 mdash
94 degC 5 sec (Figure 2) 10 ml of fresh saliva 68 degC 7 min
followed by b-Globin72 degC 7 min (Figure 3)
Saliva archived on a CPT2 mdashcollection card (Figure 2)
18rdquo punch of dried b-Globinsaliva on card (Figure 3)C elegans 100 ml Ext rrf-3 94 degC 3 min (Figure 2) mdash
25 ml Tissue Prep 35 cycles of 1 C elegans in solution 100 ml Neut B 94 degC 30 sec
(Extraction 58 degC 1 minincubated at 72 degC 15 min55 degC for followed by 10 minutes) 72 degC 7 min
Primer Name Size Forward Sequence Reverse SequenceIL-1b(Interleukin 1 Beta)1 1181 bp 5rsquo-TCTGGGGTTGATGTAGGA-3rsquo 5rsquo-GGGCTGGAAAAATGGTC-3rsquob-Globin2 5000 bp 5rsquo-CCTCAGCCTCAGAATTTGGCA-3rsquo 5rsquo-TCTCCCCAACCTCCCCCATCTA-3rsquoCPT21 1820 bp 5rsquo-CTGTCAGCCTTACACTGACCC-3rsquo 5rsquo-AGGAAGGGAGGATGAGACGT-3rsquo(carnitine palymitoyltransferase II)
SPB2 547 bp 5rsquo-CTCGAATCCCCAGCACCCTTCATTTC 5rsquo-GAGGTGCCATGGCTGAGTCA-3rsquo(human surfactant protein B) AGA-3rsquorrf-33 1079 bp 5rsquo-CACTCTTCATCGTCGGCTTTTC-3rsquo 5rsquo-CATCGCGAAGAGTCAATGTTTG-3rsquo
1 NCBI Accession Number X049642 NCBI Accession Number U013173 Wormbase gene designation F10B57
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SummaryThe Extract-N-Amptrade Tissue PCR Kit extracts DNA suitablefor PCR in 15 minutes using a straightforward protocolWhile most commonly used with mouse tails in genotypingexperiments the kit has been shown to work with a vari-ety of starting materials including mouse tissues buccalswabs hair saliva saliva archived on a collection cardand C elegans DNA prepared with the Extract-N-AmptradeTissue PCR Kit is stable for at least 6 months at 4 degC allowingfor multiple reassays Short to medium-sized PCR targets(up to 5 kb) can be amplified from the extracts on a con-sistent basis Furthermore the PCR products are suitablefor sequencing
AcknowledgementsThe authors thank Jessica Copeland Jaime Miller andBruce Blackerby of Sigma-Aldrich Biotechnology RampD forproviding the sequence data Kevin Ray and Carol Kreaderfor their critical reading and contributions to the prepa-ration of this article Also thanks to Dave Hanson andSudhir Nayak from the Tim Schedl lab at WashingtonUniversity St Louis MO
References1 Dieffenbach Carl W and Dveksler Gabriela S (eds) PCR Primer A
Laboratory Manual p 3 (Cold Spring Harbor Laboratory Press 1995)2 Smial D et al Rapid cost-effective gene mutation screening for carni-
tine palmitoyltransferase II deficiency using whole blood on filterpaper Clinical Chemistry 45 2035-2038 (1999)
3 Lin Z and Floros J Protocol for genomic DNA preparation from freshor frozen serum for PCR amplification BioTechniques 29 460-466(2000)
Refer to Table 1 and Table 2 on next page for PCR primers used for eachsample type cycling parameters and sequences for the primers
Ordering InformationExtractions
Product Description Amplifications
XNATS REDExtract-N-Amp Tissue PCR Kit 1010
XNAT REDExtract-N-Amp Tissue PCR Kit 100100
XNATR REDExtract-N-Amp Tissue PCR Kit 10001000
XNAT2 Extract-N-Amp Tissue PCR Kit 100100
XNAT2R Extract-N-Amp Tissue PCR Kit 10001000
E 3004- Extract-N-Amp Tissue PCR mdash12 ml Reaction Mix 1000
R 4775- REDExtract-N-Amp Tissue PCR mdash12 ml Reaction Mix 1000
feature article continued
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Table 1 DNA Extraction and PCR Conditions
Table 2 Primer Size and Sequence Information
Extraction Reference to Quantitative Tissue Type Volumes Primers PCR Conditions Agarose Gel PCR ConditionsMouse Tail 100 ml Ext Il-1b 94 degC 3 min (Figure 2) 94 degC 3 min
25 ml Tissue Prep 35 cycles of 40 cycles of 05-1 cm of 100 ml Neut B 94 degC 45 sec 94 degC 30 secmouse tail tip 58 degC 1 min 58 degC 30 sec
72 degC 2 min 72 degC 15 minMouse Brain Tissue followed by (Figure 2) mdash
72 degC 7 min5 mg frozen mouse brainBuccal Swab 200 ml Ext CPT21 and CPT2 and SPB CPT2 94 degC 3 min
25 ml Tissue Prep b-Globin 94 degC 3 min (Figure 2) 40 cycles of 200 ml Neut B for standard 35 cycles of 94 degC 30 sec
PCR SPB2 for 94 degC 30 sec b-Globin 60 degC 1 minQuantitative 60 degC 1 min (Figure 3) 72 degC 2 minPCR 72 degC 2 min
Hair 100 ml Ext CPT21 and followed by CPT2 mdash25 ml Tissue Prep b-Globin 72 degC 7 min (Figure 2)
1 hair shaft root end 100 ml Neut Bin solution b-Globin 5 kb b-Globin
94 degC 5 sec (Figure 3)Saliva 45 cycles of CPT2 mdash
94 degC 5 sec (Figure 2) 10 ml of fresh saliva 68 degC 7 min
followed by b-Globin72 degC 7 min (Figure 3)
Saliva archived on a CPT2 mdashcollection card (Figure 2)
18rdquo punch of dried b-Globinsaliva on card (Figure 3)C elegans 100 ml Ext rrf-3 94 degC 3 min (Figure 2) mdash
25 ml Tissue Prep 35 cycles of 1 C elegans in solution 100 ml Neut B 94 degC 30 sec
(Extraction 58 degC 1 minincubated at 72 degC 15 min55 degC for followed by 10 minutes) 72 degC 7 min
Primer Name Size Forward Sequence Reverse SequenceIL-1b(Interleukin 1 Beta)1 1181 bp 5rsquo-TCTGGGGTTGATGTAGGA-3rsquo 5rsquo-GGGCTGGAAAAATGGTC-3rsquob-Globin2 5000 bp 5rsquo-CCTCAGCCTCAGAATTTGGCA-3rsquo 5rsquo-TCTCCCCAACCTCCCCCATCTA-3rsquoCPT21 1820 bp 5rsquo-CTGTCAGCCTTACACTGACCC-3rsquo 5rsquo-AGGAAGGGAGGATGAGACGT-3rsquo(carnitine palymitoyltransferase II)
SPB2 547 bp 5rsquo-CTCGAATCCCCAGCACCCTTCATTTC 5rsquo-GAGGTGCCATGGCTGAGTCA-3rsquo(human surfactant protein B) AGA-3rsquorrf-33 1079 bp 5rsquo-CACTCTTCATCGTCGGCTTTTC-3rsquo 5rsquo-CATCGCGAAGAGTCAATGTTTG-3rsquo
1 NCBI Accession Number X049642 NCBI Accession Number U013173 Wormbase gene designation F10B57
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Table 1 DNA Extraction and PCR Conditions
Table 2 Primer Size and Sequence Information
Extraction Reference to Quantitative Tissue Type Volumes Primers PCR Conditions Agarose Gel PCR ConditionsMouse Tail 100 ml Ext Il-1b 94 degC 3 min (Figure 2) 94 degC 3 min
25 ml Tissue Prep 35 cycles of 40 cycles of 05-1 cm of 100 ml Neut B 94 degC 45 sec 94 degC 30 secmouse tail tip 58 degC 1 min 58 degC 30 sec
72 degC 2 min 72 degC 15 minMouse Brain Tissue followed by (Figure 2) mdash
72 degC 7 min5 mg frozen mouse brainBuccal Swab 200 ml Ext CPT21 and CPT2 and SPB CPT2 94 degC 3 min
25 ml Tissue Prep b-Globin 94 degC 3 min (Figure 2) 40 cycles of 200 ml Neut B for standard 35 cycles of 94 degC 30 sec
PCR SPB2 for 94 degC 30 sec b-Globin 60 degC 1 minQuantitative 60 degC 1 min (Figure 3) 72 degC 2 minPCR 72 degC 2 min
Hair 100 ml Ext CPT21 and followed by CPT2 mdash25 ml Tissue Prep b-Globin 72 degC 7 min (Figure 2)
1 hair shaft root end 100 ml Neut Bin solution b-Globin 5 kb b-Globin
94 degC 5 sec (Figure 3)Saliva 45 cycles of CPT2 mdash
94 degC 5 sec (Figure 2) 10 ml of fresh saliva 68 degC 7 min
followed by b-Globin72 degC 7 min (Figure 3)
Saliva archived on a CPT2 mdashcollection card (Figure 2)
18rdquo punch of dried b-Globinsaliva on card (Figure 3)C elegans 100 ml Ext rrf-3 94 degC 3 min (Figure 2) mdash
25 ml Tissue Prep 35 cycles of 1 C elegans in solution 100 ml Neut B 94 degC 30 sec
(Extraction 58 degC 1 minincubated at 72 degC 15 min55 degC for followed by 10 minutes) 72 degC 7 min
Primer Name Size Forward Sequence Reverse SequenceIL-1b(Interleukin 1 Beta)1 1181 bp 5rsquo-TCTGGGGTTGATGTAGGA-3rsquo 5rsquo-GGGCTGGAAAAATGGTC-3rsquob-Globin2 5000 bp 5rsquo-CCTCAGCCTCAGAATTTGGCA-3rsquo 5rsquo-TCTCCCCAACCTCCCCCATCTA-3rsquoCPT21 1820 bp 5rsquo-CTGTCAGCCTTACACTGACCC-3rsquo 5rsquo-AGGAAGGGAGGATGAGACGT-3rsquo(carnitine palymitoyltransferase II)
SPB2 547 bp 5rsquo-CTCGAATCCCCAGCACCCTTCATTTC 5rsquo-GAGGTGCCATGGCTGAGTCA-3rsquo(human surfactant protein B) AGA-3rsquorrf-33 1079 bp 5rsquo-CACTCTTCATCGTCGGCTTTTC-3rsquo 5rsquo-CATCGCGAAGAGTCAATGTTTG-3rsquo
1 NCBI Accession Number X049642 NCBI Accession Number U013173 Wormbase gene designation F10B57
7