Genome-TALER™ Human AAVS1 Safe Harbor Gene knock
Contents
1. junction PCR assay shown below 5 F primer 3 F primer Chr 19 GO bGHpA EFla G T2A uro SV40pA HAR lt lt 5 R primer 3 R primer 1 2kb oi 15 Human AAVS1 Safe Harbor Gene knock in Kits Tech Note 1 If the 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 of the chromosomal structure modification and sequence around that region 2 One positive in junction PCR is sufficient to confirm the integration 3 Itis 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 bloodjournal hematologylibrary org content 11 7 21 5561 full html 16 Human AAVS1 Safe Harbor Gene knock in Kits VII Related Services Stable cell line services GeneCopoeia offers monoclonal stable cell line service with customized TALEN or CRISPR mediated genome modifications Cell banking service is also available TALEN CRISPR Stable Cell Line Development Services e Clone Standard cell line or Custom cell line e Standard transfection e Selection antibiotics or eGFP_ e PCR screening of stable clones e Pick single colonies Safe Harbor clone only optional e Southern blot to rule out RI e Select clones with single or double allele modifications to make cell bank e Select best2. clone with single or double modifications to make master cell bank e PCR verification for one vial of MCB Human AAVS1 Safe Harbor Gene knock in Kits Vil 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 18 Human AAVS1 Safe Harbor Gene knock in Kits VI 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
3. AL 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 precise genome engineering Human AAVS1 Safe Harbor Gene knock in Kits The GeneCopoeia Genome TALER 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 ha
4. Gen eCop e ajg Expressway to Discovery Genome TALER amp Genome CRISP Human AAVS1 Safe Harbor Gene Knock in Kits 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 301 762 0888 866 360 9531 inquiry genecopoeia com www genecopoeia com 2014 GeneCopoeia Inc Human AAVS1 Safe Harbor Gene knock in Kits USER MANUAL Genome TALER Human AAVS1 Safe Harbor Gene Knock in Kit Genome CRISP Human AAVS1 Safe Harbor Gene Knock in Kit I Introduction Il Content and Storage lll Example IV Overview of Safe Harbor Integration V Critical Steps VI Related Services VII References VIII Licensing and Warranty Statement 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 present a threat of unpredicted insertion or mutagenesis The new approach recently developed is to deliver the transgene to a predeterm
5. R primer 10uM 0 2ul Zu 50X dNTP mix 10 mM of each 2 5ul 25yl 10X PCR Reaction Buffer 21 9ul 219 Nuclease free water 0 2ul Zu Taq DNA polymerase approx 5 U ul 25ul 250ul Total volume 3 Mix the master mix very well and aliquot 24ul into each well of 96 well PCR plate or individual tubes 4 Pick each marked colony from step 1 using sterilized tips and mix in 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 5ul of the PCR reaction and run it on a 1 2 agarose EtBr gel in 1X TAE buffer to identify clones with correct insert 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 Kits Co transfection of AAVS1 genome editing tools and donor plasmid 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 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
6. SH01 d Donor in vector DC DON SH01 only 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 confluent at the time of transfection 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 5ug of each TN AAVS1 L and TN AAVS1 R 1g total and 1g 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 1g of donor HR plasmid 0 5yg 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 24 hours post transfection remove transfection media and split the
7. a Sa Ta Ra oa a bes Se ee E ES w ee ee Cem a a H i BS SS SS ES EES EES EES SS SS 2 a m e remm mee Fee Ca as Ss ae ze Figure 5 Illustration of serial dilution 2 Add 200ul cell suspension to well A1 Mix 100uI from AT with the medium in well B1 Avoid bubbles Continue this 1 2 dilution through column 1 Add 100uI of medium back to column 1 so that wells A1 through H1 contain 200ul 3 Mix cells and transfer 100uI 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 200ul 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 2x10 cells ml is a good starting concentration Increase the concentration for more difficult to grow cell lines 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 noteboo
8. 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 Begin puromycin selection 48 hours post transfection For 293T cells the recommended concentration of puromycin is 1 ug 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 5yug 5yg 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 Kits 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 100uI of medium except for well A1 which should remain empty me pe mm pm mm E wm bm pN a e Een Fo Fees re e Ces a A E E E E E ee S
9. d restriction digestion analysis or direct sequencing Recommended antibiotic concentration TN AAVS1 L Kanamycin 50ug ml TN AAVS1 R Kanamycin 50ug ml HCP AAVS1 CG02 Ampicillin 50ug ml DC DON SH01 Ampicillin 50yug ml DC RFP SH01 Ampicillin 50ug ml ORF knock in clones in DC DON SHO1 Ampicillin 50yg ml B Cloning into empty DC DON SH01 vector 1 Ligation 1 Digest and gel purify the vector plasmid Dilute it to 10ng ul 2 Setup 10ul ligation reaction for each control and test sample Volume Item 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 l 10 0 pl Total Reaction Volume 10 Human AAVS1 Safe Harbor Gene knock in Kits 3 Incubate reactions at 25 C for 1 2 hours sticky end ligation or ON at 16 C for blunt end ligation Transformation Transform competent cells transformation efficiency at least 1x10 colonies ug pUC19 with the entire ligation reaction 10ul following the provided protocol of the competent cells Plate the transformed competent cells on LB Ampicillin Carbencillin agar plates 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 1rxn 10 rxn Composition O 1p Ju 5 PCR primer 10M O 1pl Tl 3 PC
10. in kit components A AAVS1 CRISPR Cas9 and donor plasmids B Knock in verification primer pairs Additional materials required LB Agar and broth containing 50 ug ml kanamycin 6 well tissue culture plates and related tissue culture supplies Other specific media and additives specific for cell type of interest Any high transformation efficiency RecA and EndA E coli competent cells GCl 5a chemically competent E Coli Cat STK200 10 or 20 Dulbecco s Modified Eagle s Medium D MEM high glucose with sodium pyruvate and glutamine Invitrogen Cat 11995073 EndoFectin Plus Transfection Reagent Genecopoeia Cat EFP1003 01 02 Qiagen EndoFree Plasmid Maxi Kit Qiagen Cat 12362 Qiagen DNeasy Blood and Tissue Kit Qiagen Cat 69504 9 Proof 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 PONS EA D Jo Human AAVS1 Safe Harbor Gene knock in Kits lll Example bGH poly A Puro Sample Red Fluorescene Green Fluorescene Phase contrast exposure time 0 6s exposure time 0 6s exposure time 2ms Sv40 poly A Control Red Fluorescene Green Fluorescene Phase contrast expos
11. ined 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 Human AAVS1 Safe Harbor Gene knock in Kits AAVS1 locus Site specific genome editing tools ji DSB Chr ORF Donor Clone a ORF knock in AAVS1 locus J HR chr DEE E 5 GE NEDO A Analysis of GENE knocked in Figure 1 Illustration of genome editing tool mediated transgene integration at the human safe harbor AAVS1 site 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 each repeat provide a simple one to one code for binding to each DNA base in the target sequence e g NI A HD C NG T and NN GorA T
12. integration highly recommended Monoclonal master cell bank preparation and storage CRISPR Cas9 mediated AAVS1 safe harbor knockin Co transfection of AAVS1 CRISPR Cas9 and knockin clone control highly recommended Antibiotic selection or cell sorting to enrich for clones with donor integration highly recommended Isolation of single colonies Validation of HR recombinant cells Screen positive clones by junction PCR Southern blotting to eliminate clones with random donor integration highly recommended Monoclonal master cell bank preparation and storage Human AAVS1 Safe Harbor Gene knock in Kits 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 200uI 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 amplified plasmids we recommen
13. 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 for 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 additi
14. k 13 Human AAVS1 Safe Harbor Gene knock in Kits E Validation of HR recombinant cells 1 Assay for genome editing tools cutting and HR of donor vectors on samples as follows 1 AAVS1 TALENs or HCP AAVS1 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 AAVS1 CG02 donor in vector DC DON SH01 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 SH01 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 D AAVS1 homology arm 5 AAVS1 HA L and 3 AAVS1 homology arm 3 AAVS1 HA R 3 Protocol for junction PCR 1 Primer sequences Primer description Primer name Primer sequence 5 AAVS1 Posie Control Donor HOPAVSHR 5SF Se eee Forward Primer 5 AAVS1 Positive Con
15. ome TALER human AAVS1 safe harbor gene knock in kit without donor Cat SH AVS K000 Product Name Qty Concentration Shipping and Storage Shipped at room temperature TN AAVS1 L AAVS1 left TALEN 500 ng ul Stored at 20 C Shipped at room temperature AAVS1 right TALEN 500 ng yl Ge Shipped at room temperature AAVS1 donor vector 500 ng ul Stored at 20 C Shipped at room temperature DC RFP SH01 AAVS1RFP control 10 yg 500 ng l SE Ee Shipped at room temperature 5 HR primer pair 200 reactions Stored at 20 C Shipped at room temperature Stored at 20 C HQPAVSHR 3 3 HR primer pair 200 reactions 10 uM DC DON SH01 only comes with the SH AVS K100 kit AAVS1 knock in ORF donor clones can be searched from our collection of 20 000 knock in clones and purchased separately A TALEN and donor plasmids AAVS1 R E AAVS1 Left TALEN AAVS1 Right TALEN bGH poly A bGH poly A Puro AAVS1 Sv40 poly A Sv40 poly A Donor vector AAVS1 HA Left AAVS1 HA Right BAST HAL oft AAVS1 HA Right Human AAVS1 Safe Harbor Gene knock in Kits B Knock in verification PCR primers 5 F pri 3 F primer aa Bett Chr 19 Giel bGHpA EFla 5 R primer 3 R primer 1 2kb eil Figure 2 AAVS1 safe harbor gene knock in kit components A TALEN and donor plasmids B Knock in verification primer pairs Acknowledgement Design of the AAVS1 left TALEN AAVS1 right TALEN and AAVS1 donor control vectors
16. onal 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 for a particular purpose GeneCopoeia is committed to providing our customers with high quality products If you should have any questions or concerns about any GeneCopoeia products please contact us at 301 762 0888 2014 GeneCopoeia Inc For Research Use Only 2014 GeneCopoeia Inc Trademark Genome TALER Genome CRISP SH 051214 EndoFectin GeneCopoeia GeneCopoeia Inc 19
17. rbor 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 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 design Human AAVS1 Safe Harbor Gene knock in Kits ll Contents and Storage Genome TALER human AAVS1 safe harbor gene knock in kit Cat SH AVS K100 Gen
18. trol Donor HQPAVSHR 5R See datasheet Reverse Primer 3 AAVS1 Positive Control Donor 3 HQPAVSHR 3F See datasheet Forward Primer 3 AAVS1 Positive Control Donor Reverse Primer HQPAVSHR 3R See datasheet The primers are provided as mixes F R primers at 10uM 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 14 Human AAVS1 Safe Harbor Gene knock in Kits manufacturer b Perform junction PCR PCR reaction below Reagent TALEN or CRISPR Cas9 cut Positive control positive control donor donor only Genomic ane mp DNA 60 100ng l H H 10uM 5 or 3 AAVS1 PCR 4 Primer Mix H tpl 5X UltraPF Buffer Mo free 5ul 10 mM dNTPs 0 5ul 0 5ul 20mM MgSOz 2 5ul 2 5ul UltraPF 5U pl 0 25ul 0 25ul PCR grade distilled water 14 75ul 14 75ul Total 25ul 25ul 98 C 5min AAVS1 Control 98 C 20sec TALENs TALENs 3 SI EN 55 C 30sec 35 cycles ip M s 3000 2 72 C 1min Saa 72 C 7min 1500 1250 1000 750 Hold at 4 16 C Primer Set GCI 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
19. ure time 0 6s exposure time 0 6s exposure time 2ms AAVS1 Right TALEN C 5 F primer 3 F primer BE bGHpA EFia G T24 Puro SV40pA HAR lt lt 5 R primer 3 R primer en ug Figure 4 Human genome safe harbor Chr 19 D AAVS1 gene targeting Aaen Sen A AAVS1 RFP control plasmid TALENs TALENs DC RFP SHO01 800 ng was co transfected bp M g 3 g 3 with AAVS1 TALEN pair 600 ng for each or 3000 control TALEN pair into HEK293T cells in a 2500 6 well pate 2000 E B 48 hr post transfection the cells were 1000 split 1 10 into a new 6 well pate and 750 incubated in medium containing 1 0 ug ml of puromycin The images were taken Primer Set GCI after two weeks of selection C D PCR primers designed to amplify the HR junction were used to verify the specific and successful integration Human AAVS1 Safe Harbor Gene knock in Kits IV Overview of Safe Harbor Integration Plasmid propagation in E coli highly recommended Cloning into empty DC DON SH01 vector TALEN mediated AAVS1 safe harbor knockin Co transfection of AAVS1 TALEN and knockin clone control highly recommended Antibiotic selection or cell sorting to enrich for clones with donor integration highly recommended Isolation of single colonies Validation of HR recombinant cells Screen positive clones by junction PCR Southern blotting to eliminate clones with random donor
20. 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 Genome CRISP 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 All in one AAVS1 sgRNA Cas9 expression clone Shipped at room Wie SEE SEG EU temperature Stored at 20 C 10 ug 500 ng ul Shipped at room G 5 DC DON SH01 AAVS1 donor vector 10 ug 500 ng ul temperature Stored at 20 C Shipped at room temperature Stored at 20 C DC RFP SH01 AAVS1 RFP control 10ug 500 ng l 200 reactions Shipped at room WSEAS SES EES temperature Stored at 20 C 10 uM 200 reactions Shipped at room HOE ASHES a temperature Stored at 20 C 10 uM DC DON SH01 only comes with SH AVS K200 kit AAVS1 knock in ORF donor clones can be searched from our collection of 20 000 knock in clones and purchased separately Human AAVS1 Safe Harbor Gene knock in Kits A CRISPR Cas9 and donor plasmids AAVS1 sgRNA 3 FLAG NLS bGH poly A Puro bGH poly A Puro Sv40 poly A Donor vector Sv40 poly A AMP H suc on D AAVS1 HA Left Aaen HA Right DANS HALo AAVST HA Right B Knock in verification PCR primers 5 F primer 3 F primer A LE Chr 19 bGHpA EFla JFF T2A Puro SV40pA HAR so e 5 R primer 3 R primer 1 2kb P Figure 3 AAVS1 safe harbor gene knock
Download Pdf Manuals
Related Search