Genome-TALER™ Human AAVS1 safe harbor gene
Contents
1. 1 2kb M Tech Note 1 If the 3 junction PCR band is weaker than 5 junction PCR band it is likely that the amplification efficiency for the 3 junction region is lower due to the nature ofthe chromosomal structure modification and sequence around that region 2 One positive in junction PCR is sufficient to confirm the integration 3 Though rare it is possible that random integration can coexist with AAVS1 specific integration Southern blotting can be used to detect coexisting random integration The method is described in http www bloodjournal org content 117 21 5561 VI References 1 Zou J et al 2009 Gene targeting of a disease related gene in human induced pluripotent stem and embryonic stem cells Cell Stem Cell 2009 Jul 2 5 1 97 110 2 Sadelain M et al 2011 Safe harbours for the integration of new DNA in the human genome Nat Rev Cancer 2011 Dec 1 12 1 51 8 3 van Rensburg R et al 2013 Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hepatopoietic stem cells Gene Therapy 2013 20 2 201 14 4 Papapetrou EP et al 2011 Genomic safe harbors permit high amp globin transgene expression in thalassemia induced pluripotent stem cells Nat Biotechnol 2011 29 1 73 8 5 Lombardo A et al 2011 Site specific integration and tailoring of cassette design for sustainable gene transfer Nat Methods 2011 8 10 861 9 16 Human AA2. 0 2 ul 2 pl 50X dNTP mix 10 mM of each 2 5 ul 25 yl 10X PCR Reaction Buffer 21 9 ul 219 ul Nuclease free water 0 2 ul 2 ul Taq DNA polymerase approx 5 U ul 25 ul 250 yl Total volume 3 Mix the master mix very well and aliquot 24 ul into each well of 96 well PCR plate or individual tubes 4 Pick the each marked colony from step 1 using sterilized tips and mix it to each well or tube 5 Proceed with PCR using the following program 94 C 4 min 1 cycle 94 C 0 5 min then 68 C 1 min 1 kb 25 cycles 68 C 3 min 1 cycle Depending on the size of final PCR product use a shorter or longer time 6 Take 5yl of the PCR reaction and run it on a 1 2 agarose EtBr gel in 1X TAE buffer to identify clones with correct insert 4 Inoculate a positive colony containing insert in an appropriate amount of LB Ampicillin Carbenicillin broth Incubate at 37 C overnight Extract and purify the construct using an endotoxin free plasmid purification kit Sequence verification of the insert is optional 11 Human AAVS1 Safe Harbor Gene knock in Kit C Co transfection of AAVS1 genome editing tools and donor plasmid 1 Plate 100 000 to 300 000 cells well in a 6 well plate following the recommended conditions for cell type s being transfected Include wells for the following On the day before transfection trypsinize and count the cells The number of cells plated in each well should be determined so that they are 70 80 conflue
3. Gen eCop 5 niai Expressway to Discovery Genome TALER amp Genome CRISPR Human AAVS1 Safe Harbor Gene Knock in Kit Catalog SH AVS K100 Catalog SH AVS K000 Catalog SH AVS K200 Catalog SH AVS K002 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 Genome TALER Human AAVS1 Safe Harbor Gene Knock in Kit Genome CRISP Human AAVS1 Safe Harbor Gene Knock in Kit JotrogdOeHgn E 3 IlGontent and Storage E 5 MR csceE E 8 IV Overview of Safe Harbor Integration ssssssseeeneeeeemeenen ener 9 Ve ge le 10 MI Ke 16 VIL Related Services EE 17 VIII Limited Use License and Warranty ssssssseeeeeenm emen nennen 18 Human AAVS1 Safe Harbor Gene knock in Kit I Introduction Safe gene targeting Genome modification by insertion of genes of interest and other genetic elements in unique site s of chromosome s is of great value for cell engineering The genetically modified cells are valuable for therapeutic research gene function study as well as lineage tracking and analysis All these applications depend on the reliable and predictable function of the transgene without perturbing any endogenous gene and or other regulation element Random integration of the transgene on the contrary can prese
4. amplified plasmids we recommend restriction digestion analysis or direct sequencing TN AAVS1 L Kanamycin 5Oug ml TN AAVS1 R Kanamycin 5Oug ml HCP AAVS1 CG02 Ampicillin 5Oug ml DC DON SH01 Ampicillin 50yug ml DC RFP SH01 Ampicillin 50yug ml ORF knock in clones in Ampicillin 50yug ml DC DON SH01 B Cloning into empty DC DON SH01 vector 1 Ligation 1 Digest and gel purify the vector plasmid Dilute it to 10ng ul 2 Set up 10ul ligation reaction for each control and test sample 1 0 ul Digested DC DON SH01 empty vector 7 0 ul DNA insert 30 50 ng or water control 1 0 ul 10X T4 DNA ligase buffer 1 0 ul T4 DNA Ligase 40 U ul 10 0 pl Total Reaction Volume 10 Human AAVS1 Safe Harbor Gene knock in Kit 3 Incubate reactions at 25 C for 1 2 hours sticky end ligation or O N at 16 C for blunt end ligation 2 Transformation Transform competent cells transformation efficiency at least 1x10 colonies u g pUC19 with the whole ligation reaction 10ul following the provided protocol of the competent cells Plate the transformed competent cells on LB Ampicillin Carbencillin agar plates 3 Screening correct clones 1 Depending on the ratio of colony numbers for the cDNA sample vs the negative control sample randomly mark 5 or more well isolated colonies 2 Prepare a PCR Master Mix with PCR primers flanking the insert ptm tore Composition 0 1 ul 1 ul 5 PCR primer 10 uM 0 1 ul 1 ul 3 PCR primer 10 uM
5. design ll Content and storage Genome TALER human AAVS1 safe harbor gene knock in kit Cat SH AVS K100 Genome TALER human AAVS1 safe harbor gene knock in kit without donor Cat SH AVS K000 Shipped at room temperature TN AAVS1 L AAVSI left TALEN 10 ug 500 ng ul E DIO TN AAVS1 R AAVS1 right TALEN 10 ug 500 ng ul EAE ei reem tampereiure Stored at 20 C DC DON SH01 AAVS1 donor vector 10 ug 500 ng ul Shipper ek room tampercitre Stored at 20 C DC RFP SH01 AAVS1 RFP control 10 500 ng ul SE i Hg HH Stored at 20 C y 200 Shipped at room temperature HQPAVSHR 5 5 HR primer pair CSI 10 uM Stored at 20 C 3 200 Shipped at room temperature HQPAVSHR 3 3 HR primer pair EE 10 uM Stored at 20 C DC DON SH01 only comes with SH AVS K100 kit AAVS1 knock in ORF donor clones can be customized and purchased separately Acknowledgement Design of the AAVS1 left TALEN AAVS1 right TALEN and AAVS1 donor control vectors was performed by Dr Jizhong Zou of the NIH Center for Regenerative Medicine a Common Fund initiative of the U S National Institutes of Health Human AAVS1 Safe Harbor Gene knock in Kit A TALEN and donor plasmids _aavsrr 0 AAVS1 Right TALEN AAVS1 Left TALEN Sv40 poly A Sv40 poly A AAVS1 HA Left AAVS1 HA Right AAVS1 HA Left AAVS1 HA Right B Knock in verification PCR primers 5 F primer 3 F primer Chr 19 bGHpA EFla GFF T2A Puro u lt 5 R primer 3
6. 2 Add 200uI cell suspension to well A1 Mix 100jl from AT with the medium in well B1 Avoid bubbles Continue this 1 2 dilution through column 1 Add 100pl of medium back to column 1 so that wells A1 through H1 contain 200ul 3 Mix cells and transfer 100yl of cells from column 1 into column 2 Mix by gently pipetting Avoid bubbles Repeat these 1 2 dilutions through the entire plate Bring the final volume to 200 ul by adding 100ul of medium to all but the last column of wells 4 Incubate plates undisturbed at 37 C 5 Cells will be observable via microscopy over 3 days and be ready to score in 5 8 days depending on the growth rate of cells Mark each well on the cover of the plate indicating which well contains a single colony These colonies can later be subcultured from the well into larger vessels Tech Note 1 Adding 4000 cells in well A1 2X 104 cells ml is a good starting concentration Increase the concentration for more difficult to grow cell lines 13 Human AAVS1 Safe Harbor Gene knock in Kit 2 If the reporter gene is fluorescent determine which of these colonies express it If the reporter gene is not observable you will have to wait until later in the culture process 3 Label each well with a single colony using a unique identification number and record this number on the plate and in your notebook E Validation of HR recombinant cells 1 Assay for genome editing tools cutting and HR of donor vectors on samples a
7. R primer 1 2kb P Figure 2 Genome TALER human AAVS1 safe harbor gene knock in kit components A AAVS1 TALEN and donor plasmids B Knock in verification primer pairs Genome CRISPTM human AAVS1 safe harbor gene knock in kit Cat SH AVS K200 Genome CRISP human AAVS1 safe harbor gene knock in kit without donor Cat SH AVS K002 Prts AAVST Shipped at room temperature HCP AAVS1 CG02 sgRNA Cas9 10 ug 500 ng ul COE EU i expression clone 2 AAVS1 donor Shipped at room temperature De DON SIC vector 10 pg 500 ng pl Stored at 20 C DC RFP SH01 AAVS1 RFP control 10 ug 500 ng ul SE Stored at 20 C 200 Shipped at room temperature HQPAVSHR 5 5 HR primer pair EE 10 uM Stored at 20 C HOPAVSHR 3 3 HR primer pair 200 10 uM Shipped at room temperature Stored at 20 C DC DON SH01 only comes with SH AVS K200 kit AAVS1 knock in ORF donor clones can be customized and purchased separately reactions Human AAVS1 Safe Harbor Gene knock in Kit A CRISPR Cas9 and donor plasmids AAVS1 sgRNA 3 FLAG NLS AAVS1 sgRNA Cas9 clone bGH poly A Puro bGH poly A Puro AAVS1 Sv40 poly A Sv40 poly A Donor vector AAVS1 HA Left AAVS1 HA Right AAVS1 HA Left AAVS1 HA Right B Knock in verification PCR primers 5 F primer 3 F primer Chr 19 bGHpA EFla GEI T2A Puro SV40pA HAR a a 5 R primer 3 R primer 1 2kb e Figure 3 Genome CRISP human AAVS1 safe harbor gene knock
8. VS1 Safe Harbor Gene knock in Kit VII Related Services Stable cell line services GeneCopoeia offers monoclonal stable cell line service with customized TALEN or CRISPR Cas9 mediated genome modifications Cell banking service is also available TALEN CRISPR Stable Cell Line Development Services e f DNA RNA Standard cell line or Custom cell line kaen _ Standard transfection i e Selection antibiotics or eGFP 4a 4n e Pick single colonies Select clones with single or double allele modifications to make cell bank ea e Select best clone with single or double modifications to make master cell bank e PCR verification for one vial of MCB 17 Human AAVS1 Safe Harbor Gene knock in Kit VIII Limited Use License and Warranty Limited Use License Following terms and conditions apply to use of the Genome TALER human AAVS1 Safe Harbor Gene Knock in Kit amp Genome CRISP human AAVS1 Safe Harbor Gene Knock in Kit the Product 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 modified fo
9. ach repeat appears to provide a simple one to one code for binding to each DNA base of in the target sequence e g NI A HD C NG T and NN G or A TAL effectors have been utilized to create site specific gene editing tools by fusing target sequence specific TAL effectors to nucleases TALENS transcription factors TALE TFs and other functional domains These fusion proteins can recognize and bind chromosome target sequences specifically and execute their gene editing functions such as gene knockout knockin with donor plasmid modification activation repression and more Introduction to CRISPR Cas9 In the CRISPR Cas9 system the complex of a CRISPR RNA crRNA annealed to a trans activating crRNA tracrRNA is sufficient to guide the Cas9 endonuclease to a specific genomic sequence to generate a double strand break DSB in the target DNA This system can be simplified by fusing crRNA and tracrRNA sequences to produce a synthetic chimeric single guided RNA sgRNA The selected target sequence consists of a 20 bp DNA sequence complementary to the crRNA or the chimeric sgRNA followed by the trinucleotide 5 NGG 3 protospacer adjacent motif PAM which is recognized by the Cas9 and essential for cleavage This RNA guided DNA recognition mechanism of CRISPR Cas9 provides a simple but powerful tool for precision genome engineering The GeneCopoeia Genome TALER human AAVS1 safe harbor gene knock in kit is designed to specifical
10. ia and split the cells 1 10 and 1 20 in complete growth media w antibiotics Plate cells into 6 well plates and save a set of plate s for characterization of samples by junction PCR assay see below Allow cells to recover for 24 hours 4 Begin puromycin selection 48 hours post transfection For Neuro2A cells the recommended concentration of puromycin is 1 pg ml Tech Note Establishing a kill curve on untransfected cells can determine the effective working puromycin concentration for a target cell line The concentration of puromycin typical working range of 0 5pg 5p g ml that kills gt 90 of cells after 48hours of selection is the correct dose for the cells being selected 12 Human AAVS1 Safe Harbor Gene knock in Kit D Clonal isolation of cell lines Serial dilution is widely used to isolate single clones with desired modifications followed by an expansion period to establish a new clonal cell line Like most clonal isolation methods there is no guarantee that the colonies arose from single cells A second round is advised to increase the likelihood of clonal isolation Also it is worth noting that cell types can vary substantially in their responses to single cell isolation therefore literature specific to the cell type of interest should be consulted 1 Fill each well of a sterile 96 well plate with 100 ul of medium except for well A1 which should remain empty sEEEEEZEZEE Figure 6 Illustration of me dilution
11. in kit components A AAVS1 CRISPR Cas9 and donor plasmids B Knock in verification primer pairs Additional materials required 1 LB Agar and broth containing 50 ug ml Ampicillin 2 6 well tissue culture plates and related tissue culture supplies 3 Other specific media and additives specific for cell type of interest A Am high transformation efficiency RecA and EndA E coli competent cells GCI 5a chemically competent E Coli Cat STK200 10 or 20 5 Dulbecco s Modified Eagle s Medium D MEM high glucose with sodium pyruvate and glutamine Invitrogen Cat 11995073 6 EndoFectin Plus Transfection Reagent Genecopoeia Cat EFP1003 01 02 7 Qiagen EndoFree Plasmid Maxi Kit Qiagen Cat 12362 8 Qiagen DNeasy Blood and Tissue Kit Qiagen Cat 69504 9 iProof High Fidelity DNA Polymerase BioRad Cat 172 5301 10 Fetal Bovine Serum Invitrogen Cat 16000036 11 Penicillin Streptomycin Invitrogen Cat 15070063 12 Trypsin EDTA Sigma Cat T3924 13 Optional For difficult to transfect cells the use of an electroporation system e g Lonza s NucleoFector or Invitrogen s Neon system is highly recommended Human AAVS1 Safe Harbor Gene knock in Kit Ill Example A B Sample Wi poly A Puro Red Fluorescene Green Fluorescene Phase contrast exposure time 0 6s exposure time 0 6s exposure time 2ms gt r a e gt a Control AAVS1 HA Left Red Fluorescene Green Fluoresce
12. ion or cell sorting to enrich for clones with donor to enrich for clones with donor integration highly recommended integration highly recommended Isolation of single colonies Isolation of single colonies Validation of HR recombinant Validation of HR recombinant cells Screen positive clones by cells Screen positive clones by junction PCR junction PCR Southern blotting to eliminate Southern blotting to eliminate clones with random donor clones with random donor integration highly recommended integration highly recommended Monoclonal master cell bank Monoclonal master cell bank preparation and storage preparation and storage Human AAVS1 Safe Harbor Gene knock in Kit V Critical Steps A Plasmid propagation We recommend propagating the plasmids provided in the safe harbor kit before the gene targeting experiment Plasmids can be transformed using standard conditions suitable in any RecA and EndA E coli competent cell For transformation of kit plasmids we suggest plating 50 200 ul of transformed cells on fresh LB plates with relevant antibiotics Incubate the plates at 37 C overnight Inoculate colonies from the transformation and grow them at 37 C overnight in 200ml of LB media containing relevant antibiotics Use an endotoxin free plasmid DNA maxiprep kit to extract plasmid DNA after overnight growth See the table below for resistance and recommended antibiotic concentration for each plasmid To confirm integrity of the
13. ly transfer your gene of interest selection marker or other genetic element from a donor plasmid into the AAVS1 safe harbor site on human chromosome 19 via TALEN mediated homologous recombination HR HR is a natural DNA repair mechanism that occurs in response to DNA double strand breaks DSB This DSB is created by a pair of AAVS1 specific TALENS The GeneCopoeia Genome CRISP human AAVS1 safe harbor gene knock in kit is designed to specifically transfer your gene of interest selection marker or other genetic element from a donor plasmid into the AAVS1 safe harbor site on human chromosome 19 via CRISPR Cas9 mediated homologous recombination HR HR is a natural DNA repair mechanism that occurs in response to DNA double strand breaks DSB This DSB is created by an AAVS1 specific CRISPR Cas9 system Human AAVS1 Safe Harbor Gene knock in Kit Advantages Safe integration Designated AAVS1 human genome safe harbor integration site ensures transcription competency of the transgenes and presents no known adverse effect on cells Specific targeting TALEN or CRISPR mediated DNA DSBs at the AAVS1 site stimulate homologous recombination dramatically for transgene integration Single copy number Single copy number of the transgene ensures predictable expression levels simplifies phenotype interpretation and prevents transgene silencing Compatible knock in ORFs Over 20 000 sequence verified human ORFs are compatible for transgene donor DNA
14. ne Phase contrast exposure time 0 6s exposure time 0 6s exposure time 2ms SE AAVS1 AAVS1 Left TALEN Right TALEN bGH poly C 5 F primer 3 F primer Chr 19 GOI bGHpA EF1a GFP T2A Puro SVADpA HAR 5 R primer 3 R primer et D Figure 4 Human genome safe harbor AAVS1 gene targeting AAVS1 Control A AAVS1 RFP control plasmid DC RFP SH01 TALENs TALENs 800 ng was co transfected with AAVS1 TALEN pair 600 ng for each or control TALEN bp M E 3 5 B pair into HEK293T cells in a 6 well pate 3000 2500 B 48 hr post transfection the cells were split 2000 1 10 into a new 6 well pate and incubated in 1500 medium containing 1 0 ug ml of puromycin The Aen images were taken after two weeks of selection 1000 750 C D PCR primers designed to amplify the HR SU successful integration Primer Set GCI junction were used to verify the specific and Human AAVS1 Safe Harbor Gene knock in Kit IV Overview of Safe Harbor Integration Plasmid propagation in E coli highly recommended Cloning into empty DC DON SH01 vector Optional TALEN mediated AAVS1 safe CRISPR Cas9 mediated AAVS 1 harbor knockin safe harbor knockin Co transfection of AAVS1 ALEN Co transfection of AAVS1 and knockin clone control highly CRISPR Cas9 and knockin clone recommended control highly recommended Antibiotic selection or cell sorting Antibiotic select
15. nt a threat of unpredicted insertion or mutagenesis The new approach recently developed is to deliver the transgene to a predetermined and safe site in a genome AAVS1 also known as the PPP1R2C locus on human chromosome 19 is a well validated safe harbor to host a DNA fragment with expected function It has an open chromatin structure and is transcription competent Most importantly there is no known adverse effect on the cell resulting from the inserted DNA fragment of interest The GeneCopoeia AAVS1 specific TALEN or CRISPR Cas9 system can generate a DNA double strand break DSB in AAVS1 on human chromosome 19 stimulating natural DNA repair mechanisms In the presence of AAVS1 ORF knockin clones homologous recombination HR occurs resulting in integration of the DNA fragment from the ORF knockin clone into the safe harbor locus gt AAVS1 locus Site specific genome editing tools ll DSB Chr ORF Donor Clone Eo fo Ee ORF knock in AAVS1 locus J HR chr ER Eech C Analysis of GENE knocked in Figure 1 Illustration of genome editing tool mediated transgene integration at the human safe harbor AAVS1 site Human AAVS1 Safe Harbor Gene knock in Kit Introduction to TALEN Transcription activator like TAL effectors can recognize and bind host plant promoter sequences through a central repeat domain consisting of a variable number of 34 amino acid repeats The residues at the 12th and 13th positions of e
16. nt at the time of transfection a AAVS1 TALENs or HCP AAVS1 CG02 positive control DC RFP SH01 b Positive control DC RFP SH01 only c AAVS1 TALENs or HCP AAVS1 CG02 donor in vector DC DON SH01 d Donor in vector DC DON SHO01 only 2 The next day prepare transfection complexes of genome editing tool plasmids and donor plasmids using suitable transfection reagents according to the manufacturer s instructions Leave the transfection complexes on the cells to react for gt 6 hours Example For HEK293T cells using EndoFectin Plus Transfection Reagent transfect 0 5yg of each TN AAVS1 L and TN AAVS1 R 1g total and 1yg of donor vector Tech Notes 1 Since transfection efficiencies vary across different cell lines we recommend optimizing the input of genome editing tool plasmids to donor vectors for best results We recommend starting with a 1 1 ratio e g 1ug of donor HR plasmid 0 5ug of each TALEN plasmid or 1ug of HCP AAVS1 CG02 plasmid 2 For optimal results we recommend complexing DNA with transfection reagent in serum and antibiotic free media and cells growing in complete media e g DMEM F12 10 FBS w o antibiotics 3 For hard to transfect cells e g primary stem hematopoietic it may be advisable to utilize a non passive transfection method Please follow recommended guidelines provided by the manufacturer for the specific cell type s being transfected 3 24 hours post transfection remove transfection med
17. ntrol Donor Reverse Primer HQPAVSHR 3R See datasheet 14 Human AAVS1 Safe Harbor Gene knock in Kit The primers are provided as mixes F R primers at 10M Validation of either the 5 or 3 homology arms for donor integration is usually sufficient however both arms can be done for additional confirmation 2 Protocol details for junction PCR assay a Isolate genomic DNA from positive control cells or test sample cells using a suitable genomic DNA miniprep kit Please follow the protocol recommended by the manufacturer b Perform junction PCR PCR reaction below TALEN cut positive Positive control Reagent control donor donor only Genomic DNA 60 100ng ul ul ul 10u M 5 or 3 AAVS1 PCR Primer Mix 1ul 1ul 5 XUItraPFTM Buffer Mg2 free 5ul 5ul 10 mM dNTPs 0 5ul 0 5ul 20mM MgSOA4 25ul 25ul UltraPF 5U u I 0 2511 0 251 PCR grade distilled water 14 75 ul 14 75 ul Total 25u1 25u1 AAVS1 Control TALENs TALENs 98 C 5min bp M 5 3 5 B 98 C 20sec 3000 2500 55 C 30sec 35 cycles 2000 72 C 1min 1500 1250 72 C Tmin Ets 1000 Hold at 4 16 C 750 Primer Set GCI 15 Human AAVS1 Safe Harbor Gene knock in Kit Run the PCR reaction on a 1 Agarose EtBr gel in 1X TAE buffer to confirm the Junction PCR result Sample results for 5 and 3 Junction PCR Assay shown below 5 F primer 3 F primer Chr 19 Lief bGHpA EFla 3 2 Puro SV40pA HAR x 5 R primer 3 R primer 1 1kb
18. r resale or used to manufacture commercial products or deliver information obtained in service without prior written consent from GeneCopoeia This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research 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 c
19. s follows 1 AAVS1 TALENs or HCP AAVS 1 CG02 positive control DC RFP SH01 Select cells in Puromycin for 7 10 days The resulting colonies should be RFP amp GFP positive 2 Positive control DC RFP SHO01 only Select cells in Puromycin for 7 10 days after which very few colonies if any should be seen compared with Sample a The presence of PuroR RFP GFP colonies indicates random integration events 3 AAVS1 TALENs or HCP AAVS 1 CG02 donor in vector DC DON SHO01 Select cells in Puromycin for 7 10 days after which colonies should be GFP positive Expression of the insert may be detected by qPCR or Western blot 4 Donor in vector DC DON SHO01 only Select cells in Puromycin for 7 10 days after which very few colonies if any should be seen compared with Sample c The presence of PuroR GFP colonies indicates random integration events 2 To confirm donor vector integration specifically at the AAVS1 target locus junction PCR can be performed using PCR primer pairs that flank the 5 AAVS1 homology arm 5 AAVS1 HA L and 3 AAVS1 homology arm 3 AAVS1 HA R 3 Protocol for Junction PCR 1 Primer sequences IN Primer Primer description Primer name sequence 5 AAVS1 AAVS1 Positive Control Donor Forward HQPAVSHR 5F See datasheet Primer 5 AAVS1 Positive Control Donor Reverse Primer HQPAVSHR 5R See datasheet 3 AAVS1 Positive Control Donor Forward Primer HQPAVSHR 3F See datasheet 3 AAVS1 Positive Co
20. ustomers with high quality products If you should have any questions or concerns about any GeneCopoeia products please contact us at 301 762 0888 2015 GeneCopoeia Inc For Research Use Only 2015 GeneCopoeia Inc Trademark Genome TALER Genome CRISP M SH 012315 EndoFectin GeneCopoeia GeneCopoeia Inc 18
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