IndelCheckTM CRISPR/TALEN insertion or deletion
Contents
1. In addition to the steps shown in the protocol overview we also provide brief instructions for PCR primer design and genomic DNA extraction If you stop before completing all the steps please store your PCR or digestion products at 20 C until the next step Avoid repeated freeze thaw cycles We recommend that first time users perform a positive control PCR reaction using the control reagents in the kit This PCR product can serve as a control for denaturation and re annealing as well as mismatch digestion steps 1 Primer Design 1 Target site PCR primers should have a Tm value of no less than 62 C 2 For optimum results the amplicon size range should be approximately 500 800bp 3 Design primers so that the CRISPR sgRNA or TALEN target site is offset from the center of the PCR product by approximately 100 bp This ensures being able to readily resolve the cleavage products on the gel 2 Sample Preparation Option 1 Genomic DNA extraction a Harvest cells no fewer than 10 cells per well b Extract genomic DNA using your method of choice or following the provided protocol of extraction kit manufacturer Make sure the concentration of genomic DNA solution is above 25 ng uL Option 2 Cell lysate preparation a Collect cells from cell culture dish 6 well plate or 96 well plate Centrifuge at 3000 rpm at 4 C for 5 min and carefully remove the supernatant b Add 300yL 1xPBS Pipette gently to suspend cells Centrifu2. mma transcript protein mrna orientation T Contig Label List SNP PlusstrandNP 0005701 forward NT_02251719 NM_001100168 1 plus strand NP 001093638 1 forward NT 022517 19 GRCh38 p2 View snp on GeneModel Clinical Source in gene region cSNP has frequency double hit refresh gene model Contig Label Contig mrna protein mrna orientation transcript snp count contig mRNA transcript GRCh38 p2 NT_022517 19 NM_000579 3 NP_000570 1 forward plus strand 259 coding MAE ete 3D Linkout Fundion dbSNP Protein Codon Amino acid y allele residue pos pos Figure 4 Example of SNP Geneview Report on NCBI Check column mRNA pos for your SNP site of interest Check column Chr position or db SNP rs cluster id for the sequence of the site of interest 19 IndelCheck M CRISPR TALEN insertion or deletion detection system VIII Limited Use License and Warranty Limited use license Following terms and conditions apply to use of the IndelCheck CRISPR TALEN insertion or deletion detection system If the terms and conditions are not acceptable the Product in its entirety must be returned to GeneCopoeia within 5 calendar days A limited End User license is granted to the purchaser of the Product The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product must not be resold repackaged or
3. All components are optimized together for the best performance The Target Site PCR kit is optimized for robust amplification of the target site With the proprietary lysis buffer included genomic DNA isolation is no longer required The kit contains the high efficiency and high fidelity SuperHeRo DNA polymerase which produces both blunt end and sticky end PCR products compatible with a variety of sequencing vectors GeneCopoeia also provides the target site PCR primer design and synthesis Go to our website for more details The T7 Endonuclease Assay kit contains T7 endonuclease I which detects and cleaves heteroduplex DNA It also contains positive controls for both target PCR and indel mismatch cleavage Advantages Complete system to simplify your CRISPR TALEN validation and knockout clone screening Robust amplification for the target site PCR No genomic DNA isolation is required Easy to use T7 endonuclease assay with optimized conditions and positive control Il Contents and Storage IndelCheck CRISPR TALEN insertion or deletion detection system ICPE 050 ICPE 200 The T7 endonuclease assay kit comes in two sizes 50 Reaction Kit Catalog No TENI 050 200 Reaction Kit Catalog No TENI 200 Quantity Content 50 reactions 200 reactions Shipping temperature Storage temperature Digestion reagents 100uL 20 C T7E 2U uL k aaa e 400yuL SEE Stable for at least 12 months 100uL 20 C 10 T7EN
4. Buff k cS LLL Control reagents 200uL 20 C Control template amp primer mi k 800uL aper Stable for at least 12 months IndelCheck M CRISPR TALEN insertion or deletion detection system The target site PCR kit comes in two sizes 50 Reaction Kit Catalog No TPCR 050 200 Reaction Kit Catalog No TPCR 200 Quantity 50 reactions 200 reactions 650 uL x 2 20 C Lysis Buffer NEW k y 650 uL x 8 dip Stable for at least 12 months 250 uL 20 C 5XPCR Buffer k aps Stable for at least 12 months 250 uL 20 C 5XEnh k anca epas Stable for at least 12 months Shipping temperature Storage temperature 10 uL 20 C 25mM dNTP k M 40 uL eee Stable for at least 12 months 12 5 uL 20 C S HeRo DNA pol k seca di du 50 uL ioo Stable for at least 12 months Important note 125 uL 20 C 20mM Mg2 k 500 uL Mad Stable for at least 12 months Store all components at 20 C upon receipt Materials required but not supplied The following materials are required but not supplied ddH20O Avoid using autoclaved H20 The recycled steam in some autoclaves can introduce contaminants that may interfere with PCR Target site PCR primers specific to target site s with Tm 60 C The primers should flank the TALEN or sgRNA target site and generate an amplicon of approximately 500 800 bp with the TALEN or sgRNA target site offset from the center by approximately100 bp Make sure the primers are specific for the
5. blue reach 2 3 of the length of the gel 10 IndelCheck M CRISPR TALEN insertion or deletion detection system 1 234 M Gene PCR Product T7 Endonuclease NROB1 429 147 282 Figure 2 T7 Endonuclease I assay Lane 1 PCR product from negative control cells digested with T7 Endonuclease I Lane 2 PCR product from negative control cells undigested Lane 3 PCR product from sample cells digested with T7 Endonuclease l Digestion yields 3 bands 1 unmodified 2 cleavage products of predicted sizes red asterisks Lane 4 PCR product from sample cells undigested Lane M 100bp plus DNA ladder M01010A NOTE See Appendix 3 for T7 endonuclease assay of control 7 Sequence verification following step 3 1 Ligate the digested AT vector backbone or digested blunt end vector backbone and PCR product according to the guidelines provided by the manufacturer of the ligation System Include a vector only ligation control 2 Transform the ligated plasmid into a competent E coli strain according to the protocol supplied with the cells Incubate the mixture overnight at 37 C 3 Pick a minimum of 48 clones to inoculate in 3 5 ml of LB ampicillin 4 Isolate the plasmid DNA from overnight cultures by using a commercial miniprep kit 5 Verify the sequence of each colony by sequencing from the AT vector backbone using the relevant primers Reference the sequencing results against the expected genomic sequence to
6. check for the presence of CRISPR sgRNA or TALEN induced NHEJ modifications Calculate the percentage of editing efficiency as no of modified clones no of total clones It is important to pick a reasonable number of clones 224 to generate an accurate approximation of modification efficiencies 11 IndelCheck M CRISPR TALEN insertion or deletion detection system VI Notes and Troubleshooting Cleavage troubleshooting Possible Causes Recommended Solution Non specific cleavage bands are observed No cleavage products observed Nonspecific nuclease activity is observed DNA bands are too weak to be observed Non specific PCR amplification Low T7 Endonuclease activity Incorrect reaction temperature Reaction time too long Poor annealing operation PCR is introducing mutations Low positive rate of modification Insufficient amount of DNA loaded on gel Do gel purification to ensure that your amplicons are single bands See Figure 4 in Appendix Optimize PCR primers on untreated genomic DNA or cell lysates Optimaze PCR condition e fnocleavage is observed for all samples including the positive control add MnCl at a final concentration of 10mM to enhance T7 Endonuclease I activity Ensure that samples are incubated at 37 C Advoid treating with T7 Endonuclease more than 1 5 hour Perform denaturalization and annealing in heated water Let the reaction cool down naturel
7. if the primers match with the template correctly Increase the annealing tempture to 0 5 C above the Tm value Check the design of PCR primers for possible non specific binding If necessary redesign primers to improve specifity Decrease the volume of polymerase to 0 2 uL 1U Dilute template 2 or more fold and repeat the PCR Decrease the volume of polymerase to 0 2 uL 1U 13 IndelCheck M CRISPR TALEN insertion or deletion detection system VII Appendix 1 Example of using IndelCheck CRISPR TALEN indel detection system to validate CRISPR sgRNA or TALEN cleavage activity A B T7 Target PCR Gene Product ae oes A NR4A 1 775 428 347 B ESRRA 791 267 524 Figure 3 T7 Endonuclease digestion products of amplicons derived from human genomic DNA Control cells should only have a larger band corresponding to the uncut genomic amplicon Sample cells transfected with indicated Cas9 sgRNA have a larger band and smaller bands corresponding to the DNA fragments resulting from the cleavage of the genomic amplicon by T7 Endonuclease I 2 Example of using gel purification to optimize cleavage of non specific amplicons Target Gene PCR Product T7 Endonuclease NROB1 429 bp 127 bp 302 bp Figure 4 Cleavage comparisom between gel purified and unpurified non specific target site PCR products Lane1 Gel extracted PCR product digested by T7 Endonuclease Lane2 Gel extracted PCR product undigeste
8. modified for resale or used to manufacture commercial products or deliver information obtained in service without prior written consent from GeneCopoeia Use of any part of the Product constitutes acceptance of the above terms Limited warranty GeneCopoeia warrants that the Product meets the specifications described in the accompanying Product Datasheet If it is proven to the satisfaction of GeneCopoeia that the Product fails to meet these specifications GeneCopoeia will replace the Product In the event a replacement cannot be provided GeneCopoeia will provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to GeneCopoeia within 30 days of receipt of the Product GeneCopoeia s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price GeneCopoeia s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty GeneCopoeia does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product fora particular purpose GeneCopoeia is committed to providing our customers with high quality products If you should have any questions or concerns about any GeneCopoeia
9. the mixture into the well of 2 agarose EtBr gel and run in TAE or TBE buffer C Also load a 100bp DNA ladder M01010A as a size reference marker in one of the adjacent wells Run the gel at 5 V cm until the bromophenol blue has migrated 2 3 of the length of the gel Gene PCR Product T7EI 520 1 control 80 330 Figure 3 T7 Endonuclease I assay result for control Lane M 100bp plus DNA ladder M01010A Lane 1 unpurificated PCR product from control undigested Lane 2 unpurificated PCR product from control digested 16 IndelCheck M CRISPR TALEN insertion or deletion detection system 4 Search for potential SNP sites using online tools Mammalian cells contain at least 2 copies of every chromosome one copy from the mother and the other from the father So although most stretches of the chromosome will be identical to one another it is possible to have some variations between them in the forms of SNPs or other base pair differences in your target region sequence Also some tumor genes such as P53 tend to have lots of mutations So even in the negative control it is possible to get T7 cutting from PCR product denaturion and reannealing This is why when designing target primers your design strategy should include avoiding SNPs in the first place We highly recommend you obtain sequence information of the target site of your cell line before primer design and any other experiment NCBI provides a tutorial for searchin
10. Gen eCop 5 niai Expressway to Discovery IndelCheck CRISPR TALEN insertion or deletion detection system Cat No ICPE 050 Cat No ICPE 200 Cat No TENI 050 Cat No TENI 200 Cat No TPCR 050 Cat No TPCR 200 User Manual GeneCopoeia Inc 9620 Medical Center Drive 101 Rockville MD 20850 USA 301 762 0888 866 360 9531 inquiry genecopoeia com www genecopoeia com 2015 GeneCopoeia Inc USER MANUAL IndelCheck CRISPR TALEN insertion or deletion detection system l arittodU ctione cnc oU rh edem mrt 3 Il Gontents and storage eee RAD Medie 4 III Protocol OVER VIC Wass tes cede b iato Pe tod T estere att dedo daten E ORDEI 6 IV Experimental Proced res o ee red EO HE eet ted eva eoe vba Pav gode tav a 7 Tee Primer deslgric itc etm epp rto liebe dur em ert ER de 7 2 Sample preparation om tutelae aude d diia a eiit 7 3 Target site PCR and product processing ssseeeee ene 8 4 Denaturation and re annealing sssseeeeene emen eene 10 5 Digest with T7 Endonuclease l sese eene 10 6 Gel analysis siete E m 10 Ta S quence vertfICation doce dui e D REPERI 11 V Notes and Troubleshooting nt i ge pg e ERE RE e ERE te ED Roe ERR Rae OSEE e LASER ARS 12 VI Appendix ettet E A att dut ees ttes tarea EET E eee stie reet hus E 14 VII Limited Use License and Warranty ssssssseeeeee em eem ennemis 20 IndelCheck M CRI
11. Homo sapiens human Gene ID 1234 updated on 17 May 2015 Summary Official Symbol CCR5 provided by HGNC Official Full Name chemokine C C motif receptor 5 gene pseudogene provided by HGNC Primary source HGNC HGNC 1606 See related HPRD 03223 MIM 601373 Genetype protein coding RefSeq status REVIEWED Organism Homo sapiens Lineage Eukaryota Metazoa Chordata Craniata Vertebrata Euteleostomi Mammalia Eutheria Euarchontoglires Primates Haplorrhini Catarrhini Hominidae Homo Also known as CKR5 CCR 5 CD195 CKR 5 CCCKR5 CMKBR5 IDDM22 CC CKR 5 Summary This gene encodes a member of the beta chemokine receptor family which is predicted to be a Variation See variants in ClinVar See studies and variants in dbVar See Variation Viewer GRCh37 p13 E Genotypes See SNP Geneview Report See 1000 Genomes Browser GRCh37_p13 Table of contents Summary Genomic context Genomic regions transcripts and products Bibliography Phenotypes HIV 1 interactions Pathways from BioSystems Interactions General gene information Markers Clone Names Homology Gene Ontology General protein information NCBI Reference Sequences RefSeq 18 IndelCheck M CRISPR TALEN insertion or deletion detection system SNP linked to Gene genelD 1234 Via Contig Annotation GRENJO lelpdesk Send rs on all gene models to Batch Query Download all rs to file ee t Total gene model contig mRNA transcript
12. SPR TALEN insertion or deletion detection system I Introduction CRISPR TALEN introduced double stranded breaks DSBs at desired target sites can be repaired by nonhomologous end joining NHEJ which is error prone and typically results in small insertions or deletions indels near the DSB The target region is PCR amplified and the PCR products are denatured and re annealed to allow mismatched DNA to form such as wildtype indel mutation mismatches or indel mutation 1 indel mutation 2 mismatches The re annealed PCR fragments are incubated with T7 endonuclease which recognizes and cleaves mismatched DNA If two shorter bands of the predicted size are generated it usually means that CRISPR TALEN has successfully introduced indel mutations at the targeted chromosomal site Cell population GOGO GD GOGO Genomic DNA amplification TALEN CRISPR induced NHEJ Q TALEN CRISPR DSB Indel mutations V Denature amp re anneal T7 endonuclease cleavage Mismatch cleavage Figure 1 CRISPR or TALEN functional validation using the mismatch cleavage assay IndelCheck CRISPR TALEN insertion or deletion detection system The IndelCheck CRISPR TALEN insertion or deletion detection system ICPE 050 ICPE 200 is a complete system designed to simplify your validation or screening workflow It includes both the Target Site PCR kit TPCR 050 TPCR 200 and the T7 Endonuclease Assay kit TENI 050 TENI 200
13. d Lane3 Unpurified PCR product with non specific bands digested by T7 Endonuclease Lane4 Unpurified PCR product with non specific bands undigested Lane5 100bp plus DNA ladder M01010A 14 IndelCheck M CRISPR TALEN insertion or deletion detection system 3 Procedures for control target PCR and T7 endonuclease assay using the control template amp primer mix 1 Control PCR a Prepare control PCR using the following system 5 x PCR Buffer 5 pL 5 x Enhancer optional 5 wl Control template amp primer mix 4 uL 25mM dNTP 0 2 uL SuperHeRo DNApolymerase 5U ul 0 25 pL 20mM Mg2 25 uL ddH20 to25 pL Final 25 pL b Proceed with PCR using the following program 94 C 94 C 58 C 12 C 12 C 5 min 1 cycle 30s 30s 35 cycs 1 min 5 min 1 cycle 2 Denaturation and re annealing a For PCR products amplified with GeneCopoeia target PCR kit combine the following Unpurified PCR product 200 500 ng 5 19uL Nuclease free water Add upto 19 uL Total 19 uL 15 IndelCheck M CRISPR TALEN insertion or deletion detection system b Mixand centrifuge for a few seconds c Heatat95 C 5 minutes d Reanneal by allowing the denatured PCR products cool down to RT 3 Digest with T7 Endonuclease a Add 1uL of 2U uL T7 Endonuclease I b Incubate at 37 C for 20 60 minutes 4 Gel analysis a Add 1 10 volume of 10 X loading buffer with 0 1 SDS to each reaction and mix thoroughly b Load half of
14. g for SNP information of a gene We offer a briefdescription of the process here eBY GENE NAME 1 Search the Gene database with the gene name If you know the gene symbol and species enter them as follows tpo sym AND human orgn Resources v How To v Filters activated Current only Clear all 2 Click on the desired gene viccR5 Save search Advanced Display Settings v Tabular 20 per page Sort by Relevance Send to v Did you mean CCR5 as a gene symbol Search Gene for CCR5 as a symbol 2 Results 1 to 20 of 411 Page 1 of21 Next Last gt gt Filters activated Current only Clear all to show 417 items Name Gene ID Description Location Aliases MIN chemokine C C Chromosome 3 CC CKR 5 CCCKR5 601 ID 1234 motif receptor 5 NC 000003 12 CCR 5 CD195 CKR gene pseudogene 46370142 46376206 5 CKR5 CMKBR5 Homo sapiens IDDM22 17 human IndelCheck M CRISPR TALEN insertion or deletion detection system 3 In the list of Links on the right click GeneView in dbSNP If the link is not present no SNPs are currently linked to this gene 4 Forhuman genes another option is to go to the variation section Click on Variation in the table of contents in the upper right and follow links to Variation Viewer for either the GRCh37 hg19 or GRCh39 h38 assemblies to the 1000 Genomes Browser ClinVar and more CCR5 chemokine C C motif receptor 5 gene pseudogene
15. ge at 3000 rpm at 4 C for 5 min and remove the supernatant c Add 300uL 1xPBS and resuspend cells Sample the suspension to calculate the cell number if necessary Centrifuge at 3000 rpm at 4 C for 5 min and remove the supernatant as completely as possible Proceed to lyse or store the pellet at 80 C d Add 25uL Lysis Buffer and lyse cells at 65 C for 15 min then 95 C for 10 min Quickly put it on ice afterward IndelCheck M CRISPR TALEN insertion or deletion detection system NOTE The volume of Lysis Buffer can be adjusted basing on the cell number At least 50 000 and no more than 5x10 cells are recommended for use in 25 uL Lysis Buffer For confluent cells of a well of 6 well plate add 200 uL 600 uL Lysis Buffer For confluent cells of a well of 96 well plate add 50 100 uL Lysis Buffer For amplifying fragment 1 kb we suggest prolonging the cell lysis at 65 C for 40 min but no more than 1h However It is not not necessary to obtain complete cell lysis in most experiments The remaining cells can be stored at 80 C or for continued culture e Frozen centrifuge at 12000 rpm for 1 min NOTE Too much floc after centrifuge suggests too little lysis occurred After transferring the suspension to another tube the precipitate can be resuspended by adding another 25 uL Lysis Buffer f Proceeded to PCR reaction with Target PCR kit The cell lysate can be stored at 4 C for no more than one week or 20 C for several mon
16. intended site Avoid using primers that contain inosine Avoid to have potential SNP sites or sequence differences between alleles in your target region NOTE We highly recommend you to obtain sequence information of the target site of you cell line before primer design and any other experiment It is possible for mammallian cells which are usually dipoid to have sequence differences between alleles This may cause false positive results when using T7 endonuclease to digest negative controls Such sites should be avoided when designing target PCR primers See Appendix 4 for further instructions on checking potential SNP sites using online tools GeneCopoeia also provides design and synthesis services for sequence specific target site PCR primers IndelCheck M CRISPR TALEN insertion or deletion detection system IV Protocol Overview pu mmm uum m m m m sunu mai mem men mem y Genomic DNA Y Cell lysis extraction Optional IL KE E LL Q o xe c f O Q ES O O xe e O t Denaturation and Ligate to AT re annealing vectors T7 Endonuclease Sequence verify digestion modifications Electrophoresis and analysis IndelCheck M CRISPR TALEN insertion or deletion detection system IV Experimental Procedures This section provides instructions for validating CRISPR sgRNA or TALEN chromosomal cleavage activity using the IndelCheck CRISPR TALEN insertion or deletion detection system
17. ize We strongly recommend use of a high fidelity DNA polymerase to reduce the amount of base misincorporation during PCR and subsequent false positives The PCR product can be directly used as the substrate for T7 Endonuclease digestion IndelCheck M CRISPR TALEN insertion or deletion detection system 2 Purification or gel extraction of correct sized band from non specific PCR background See Appendix 2 for using gel purification to optimize cleavage of non specific amplicons 4 Denaturation and re annealing 1 For purified genomic PCR product DNA gt 25ng uL 200 500 ng 10X T7EN Buffer 2 yL Nuclease free water Add upto 19 uL Total 19 uL For PCR products amplified with GeneCopoeia target PCR kit combine the following Unpurified PCR product 200 500 ng 5 19uL Nuclease free water Add upto 19 pL Total 19 uL 2 Mix and centrifuge for a few seconds 3 Heat at 95 C for 5 minutes 4 Reanneal by allowing the denatured PCR products to cool down to RT 5 Digest with T7 Endonuclease 1 Add 1pL of 2U pL T7 Endonuclease I 2 Incubate at 37 C for 20 60 minutes 6 Gel analysis 1 Add 1 10 volume of 10 X loading buffer with 0 1 SDS to each reaction and mix thoroughly 2 Load half of the mixture into the well of 296 agarose EtBr gel and run in TAE or TBE buffer 3 Also load a 100bp DNA ladder M01010A as a size reference marker in one of the adjacent wells Run the gel at 5 V cm 11 V cm until the bromophenol
18. ly with the water Perform the denaturalization and annealing step in a PCR machine as follows 1 95 C 5min 2 94 C 2 C cycle 10 20 sec 3 93 C 2 C cycle 10 20sec and go to step 2 34 cycles Besure to use a high fidelity polymerase for PCR amplification If possible optimize the conditions of your genome editing experiment e g design new CRISPR sgRNA or TALENS Besure to load enough DNA to enable ready visualization on the gel Also load equal amounts of total PCR product DNA in each lane 12 IndelCheck M CRISPR TALEN insertion or deletion detection system Target site PCR troubleshooting Possible Causes Recommended Solution No expected bands Non specific bands Smear Suboptimal PCR conditions Concentration of PCR template is too low Incomplete lysis Poor PCR primer design Suboptimal PCR conditions Poor PCR primer design Too much polymerase Concentration of PCR template is too high Too much polymerase Analyze the sequence of your target If the GC content is lower than 40 do not add Enhancer in the PCR reaction Extract and purify genomic DNA to better control the template concentration Remove PBS as completely as possible before adding Lysis Buffer or the remaining PBS will dilute the lysis buffer Caluculate the cell number by hemocytometer or cellometer before lysing the cells Adjust the volume of lysis buffer according to the cell number Check
19. products please contact us at 301 762 0888 2015 GeneCopoeia Inc For Research Use Only 2015 GeneCopoeia Inc Trademark GeneCopoeia GeneCopoeia Inc IndelCheck CV 082015
20. ths until use 3 Target PCR and product processing 1 Target PCR a For PCR from extracted genomic DNA prepare a Master Mix with target PCR primers flanking the insert as follows EE 5X PCR Buffer 5X Enhancer optional genomic DNA Primers 2 uM 25mM dNTP SuperHeRo DNApolymerase 5U ul 20mM Mg2 ddH20 Final 5 5 50 200 0 2 0 25 2 5 to 25 25 uL uL ng uL uL uL uL uL uL IndelCheck M CRISPR TALEN insertion or deletion detection system b For PCR from lysate prepare a Master Mix with target PCR primers as follows EEG XN 5X PCR Buffer 5 yL 5X Enhancer optional 5 yL lysate 1 uL Forward and reverse primers 2 uM 2 ul 25mM dNTP 02 pL SuperHeRo DNApolymerase 5U uL 0 25 uL 20mM Mg2 25 uL ddH20 to25 pL Final 25 pL To avoid insufficient PCR amplification adjust the volume of cell lysate based on the cell number to ensure that no less than 2000 copies of template are present in the reaction For example for HT1080 cells which contains 2 copies of each chromosome at least 1000 lysed cells are needed ina PCR reaction To get a bright and clear band on agarose gel about 10 000 lysed cells are needed NOTE See Appendix 3 for PCR system for the control mix c Proceed with PCR using the following program 94 C 5 min 1 cycle 94 C 30s 58 C 30s 35 cycs 72 C 1 min 72 C 5 min 1 cycle NOTE PCR should produce a sufficiently high yield of a SINGLE amplified band of the correct s
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