Materials
Name | Company | Catalog Number | Comments |
pStart-K | Addgene | 20346 | |
pWS-TK6 | Addgene | 20350 | |
pKD3 | Addgene | 45604 | |
pKD46 | The Coli Genetic Stock Center, CGSC | 7634 | |
PGK-neo-bpA sequence | Addgene | 13442 | |
Human BAC clones of target genes | https://bacpac.chori.org | ||
JDS246 (Cas9-003), Mammalian codon-optimized streptococcus pyogenes Cas9-3X Flag | Addgene | 43861 | |
MLM3636, Human-gRNA-ExpressionVector with U6 promoter | Addgene | 43860 | |
pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) | Addgene | 42335 | |
sgRNA design, ZiFit | http://zifit.partners.org/ZiFiT/ | ||
Off target prediction tool CasOT | http://eendb.zfgenetics.org/casot/download.php | ||
Perl | http://www.perl.org/get.html | ||
NCBI | http://www.ncbi.nlm.nih.gov/ | ||
BLAT | https://genome.ucsc.edu/cgi-bin/hgBlat?command=start | ||
sgRNA Primer synthesis | Sigma | ||
One shot Top10 Electrocomp E. Coli Competent cells | Life Technologies | C4040-50 | |
AccuPrime Pfx SupperMix | Life Technologies | 12344-040 | |
Restriction enzymes | NEB and Life Technologies | ||
Zymoclean Gel DNA Recovery Kit | Zymo Researech | D4007 | |
DNA quick extraction buffer | Epicentre | QE0905T | |
Herculase II Fusion DNA Polymerases | Agilent Technologies | 600675 | |
Digestion buffer 2 | New England Biolab | ||
6X DNA Loading Dye | Thermo Scientific | R0611 | |
TeSR-E8 Kit for hESC/hiPSC Maintenance | Stem Cell Technologies | 05940 | |
UltraPure Agarose | Life Technologies | 16500-100 | |
50xTAE | Life Technologies | B49 | |
Essential 8 medium | Life Technologies | A1517001 | |
Dulbecco’s Phosphate Buffered Saline without Calcium and Magnesium | Life Technologies | A12856-01 | |
D-MEM/F12 with Glutamax | Life Technologies | 10565018 | |
D-MEM with Glutamax | Life Technologies | 10566040 | |
Fetal Bovine Serum-ES cell qualified | Life Technologies | 10439 | |
Knockout serum replacement | Life Technologies | 10828010 | |
2-mercaptoethanol 1000X | Life Technologies | 21985023 | |
Non Essential Amino Acid | Life Technologies | 11140050 | |
Stempro Accutase | Life Technologies | A1110501 | |
Dispase | Life Technologies | 17105-041 | |
0.25% Trypsin- EDTA solution | Life Technologies | 25200-056 | |
Geltrex | Life Technologies | 12760-013 | |
ROCK inhibitor Y-27632 | Millipore | SCM075 | |
SMC4 reagent | BD | 354357 | |
Neomycin resistant MEF | Millipore | PMEF-NL | |
Hygromycin resistant MEF | Millipore | PMEF-HL | |
G418 (Geneticin) | LifeTechnologies | 11811 | |
Hygromycin B | Life Technologies | 10687010 | |
FIAU (Fialuridine, 1-2-Deoxy-2-fluoro-ß-D-arabinofuranosyl-5-iodouracil | Moravek Biochemicals and Radiochemicals | M251 | |
Electroporator: Gene Pulser Xcell | Bio-Rad | ||
0.4 cm electroporation cuvette | Bio-Rad | 165-2088 | |
DIG-High prime DNA labeling and detection starter kit II | Roche | 11585614910 | |
Hybridization denature solution | VWR | 82021-478 | |
PCR DIG probe synthesis kit | Roche | 11636090910 | |
DIG wash set | Roche | 11585762001 | |
Anti-Digoxigenin (DIG-AP) | Roche | 11093274910 | |
CSPD chemiluminescence system | Roche | 11755633001 | |
DIG wash and block buffer set | Roche | 11585762001 | |
50X TAE buffer | Life Technologies | 24710030 | |
Blotting buffer (25 mM Tris pH 7.4, 0.15 M NaCl, 0.1% Tween20) | |||
Hoefer Ultraviolet Crosslinker | Fisher Scientific | 03-500-308 | |
Spermidine | Fisher | AC13274-0010 | |
Tris HCl 2M (pH 7.5) | VWR | 200064-506 | |
Denville Scientific blue bio film 8x10 | Fisher | nc9550782 | |
DNA molecular weight marker II ( DIG-labeled ) | Roche | 11218590910 | |
Amersham Blotting membrane Hybond-N+ | Roche | 95038-400 | |
Pyrex glass drying tray | Fisher | 15-242A | |
Kimberly-Clark C-fold paper towels | Fisher | 06-666-32B | |
Whatman 3MM paper (26X41) | Fisher | 05-713-336 | |
Hybridization bag | Roche | 11666649001 | |
Hybridization tubes | Fisher | 13-247-300 | |
Hybridization oven rotisserie Shake 'n' Stack | Fisher | HBMSOV14110 |
References
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