...inhibits chain elongation. 50-500 ug/mL Puromycin pac HeLa, 293HEK, Jurkat T cells Inhibits protein synthesis...

...expression: pAC2 , pAC152 , pAC153 , pAC154 ; pmax dCas9 activator expression: pAC91 , pAC92 , pAC93 , pAC94...Fly: gRNA cloning pAc-sgRNA-Cas9 PDF 165.1 KB Marraffini Bacteria: pCas9 new spacer cloning pCas9 PDF ...pAC94 , pAC95 ; dCas9 activator gateway donors: pAC84 , pAC1 , pAC147 , pAC148 , pAC149 ; gateway destination...destination: pAC90 PDF 1.0 MB Katic Nematode: Cas9 and gRNA use Cas9 (pIK86) ; gRNA empty backbone (pDR274...PDF 125.6 KB Marraffini Bacteria: pCRISPR new spacer cloning pCRISPR PDF 106.7 KB Mendenhall & Myers Mammalian...PDF 248.6 KB Zhang sgRNA cloning lentiCRISPR v2 packaging plasmids: pVSVg , psPAX2 ; positive control: CMV-EGFP...vector system: lentiCas9-Blast and lentiGuide-Puro packaging plasmids: pVSVg , psPAX2 positive control: CMV-EGFP...

...over to our CRISPR Plasmids and Resources page.  PAC-MAN (prophylactic antiviral CRISPR in human cells...

...TagBFP, iCre CAG Mammalian Capecchi 113849 pCAGGS-pac-T2A-iCre iCre CAG Mammalian Capecchi 116879 CAG-Cremyc...Tamoxifen inducible CAG Mammalian Capecchi 125821 pCAGGS-pac-T2A-iCre (v2) iCre and PuroR CAG Mammalian Capecchi...

...BbsI none S. pyogenes Virmilion Bullock and Port pAc-sgRNA-Cas9 49330 Drosophila BspQI yes, cut S. pyogenes...pLKO5.sgRNA.EFS.PAC 57825 Mammalian/Lentiviral BsmBI none S. pyogenes Puro Ebert pL-CRISPR.EFS.PAC 57828...pyogenes Duchek pAC155-pCR8-sgExpression 49045 Mammalian BbsI none S. pyogenes Jaenisch pAC153-dual-dCas9VP96...vector 51132 Mammalian BsaI none S. pyogenes Huang pAC154-dual-dCas9VP160-sgExpression 48240 Mammalian BbsI...pyogenes tagRFP Ebert pFGC-pcoCas9 52256 Plant AscI, PacI, SbfI yes, cut S. pyogenes Sheen pT7-gRNA 46759 ...pDCC5 59984 Fly BbsI yes, nick S. pyogenes Duchek pAC5-dual-dCas9VP48-sgTetO 48237 Mammalian BbsI yes, ...Lentiviral BsmBI yes, cut S. pyogenes Puro Ebert pL-CRISPR.SFFV.PAC 57829 Mammalian/Lentiviral BsmBI yes, cut S...

...columns have a finite binding capacity. If your sample exceeds the capacity, divide the sample among multiple...columns. For information about the total binding capacity for your columns, refer to the manufacturer’s ...Place a mark on the side of the column where the compacted resin is slanted upward. Place column in centrifuge...centrifugation step Pro-Tip Resin will appear compacted after centrifugation. Discard the flow through.... Slowly apply the sample to the center of the compact resin bed. Pro-Tip For sample volumes <1.5 mL top...the reservoir of the column. Fill the remaining space in the column with PBS and pipette several times...through into a waste container. Fill the remaining space in the column with PBS and pipette several times...

... Timeline Day 0: Seed 293T packaging cells Day 1 (pm): Transfect packaging cells Day 2 (am): 18 h post-transfection...This procedure can be modified for alternative packaging cell lines or transfection reagents. Once produced...Considerations Before You Start The health of the packaging cell line is critical for obtaining high viral... 15 for viral production. Procedure Seed 293T packaging cells at 3.8×10 6 cells per plate in DMEM Complete...supernatant at 2100 rcf for 5 minutes to pellet any packaging cells that were collected during harvesting. Filter...

...control vector containing scrambled shRNA. psPAX2 Packaging plasmid for producing viral particles. pMD2.G ...particles. Note: pLKO.1 can also be used with packaging plasmid pCMV-dR8.2 dvpr and envelope plasmid pCMV-VSVG...tubes VWR: #87003-290 Note: pLKO.1 could also be packaged using pCMV-dR8.2 dvpr and pCMV-VSVG from the Robert...transfection: 1 μg pLKO.1 shRNA plasmid 750 ng psPAX2 packaging plasmid 250 ng pMD2.G envelope plasmid to 20 μl... may want to vary the ratio of shRNA plasmid, packaging plasmid, and envelope plasmid to obtain the ratio...

...you want to expand that with the addition of NdeI, PacI, AscI, and MfeI. With oligo overlap cloning, you...the original vector. Designing oligos To add NdeI, PacI, AscI and MfeI sites between the EcoRI and SalI ...the additional sites in tandem ( NdeI - CATATG , PacI - TTAATTAA , AscI - GGCGCGCC , MfeI - CAATTG ). ...

...Procedure The balance in the photo above has a capacity of 200 g, as indicated by the number above the...is best. Check that the balance has the correct capacity for the material that you are weighing by looking... you’re weighing out an amount larger than the capacity of your balance or weighing boat, you can weigh...

...experiment, make sure your workspace is clean and uncluttered. Disinfect your workspace with appropriate materials...times for each agent. Make sure you have enough space to work at your lab bench. It’s possible that while...

... differences, and plasmid recombination can all impact the representation of individual plasmids in the...problem as these sequences are not efficiently packaged into lentiviral particles. See below for options...overloaded. If you find that your pellet outstrips the capacity listed in the Maxiprep protocol, scale the reagent...

... appropriate for the biosafety level of the lab space Reagents Sample to be centrifuged Water Background...picture above. Wear PPE appropriate for the lab space in which you are working. Even if your samples may...

... of AAV is currently limited by its small packaging capacity (∼4.5 kb including ITRs), though there is...as well as our Packaged on Request service to give you more flexibility. AAV Packaging Plasmids The following...AAV1 AAV packaging plasmid, expressing Rep2 and Cap1 Wilson 104963 pAAV2/2 AAV2 AAV packaging plasmid,... AAV5 AAV packaging plasmid, expressing Rep2 and Cap5 Fan 112863 pAAV2/7 AAV7 AAV packaging plasmid, expressing...AAV8 AAV packaging plasmid, expressing Rep2 and Cap8 Wilson 112865 pAAV2/9n AAV9 AAV packaging plasmid,...Blog: AAV use in CRISPR Viral Vectors 101 eBook Packaging Plasmids AAV Plasmids Adeno-associated viruses...interest and effort directed toward expanding this capacity. Traditionally, AAV requires the presence of another...

...effects. The library was cloned into the Liu lab’s own pAc-sgRNA-Cas9 insect expression backbone, which expresses...be found here). To use the library, the authors packaged it into lentivirus and used the resulting lentiviral...large size of dCas9-VPR limits one’s ability to package it into lentivirus and later create stable cell...cells. The piggybac system has a much larger cargo capacity than lentivirus and is capable of efficiently ...

...along with additional packaging components Trono 63586 psPAX2-D64V 2nd Packaging For making Integrase ...22500 pNHP 2nd Packaging Contains gag, pol, tat, rev Kasparov 1732 pCPRDEnv 2nd Packaging part of the FELIX...strain). Use with packaging construct encoding Tat. Reiser 17531 pCD/NL-BH*DDD 2nd Packaging Contains Gag/...Addgene lentiviral packaging, envelope, and transfer plasmids Viral...Lentiviral Pooled Libraries All Viral Plasmids Packaging Plasmids Transfer Plasmids Lentiviruses, a subset...lentiviral terms Popular Lentiviral Envelope and Packaging Plasmids One safety feature of lentivirus used...multiple plasmids. Some plasmids (typically called packaging and envelope plasmids) encode components of the...

... Plasmid 48223 pAC91-pmax-dCas9VP64 : dCas9VP64 on pmax expression vector. 48224 pAC92-pmax-dCas9VP96 ...vector. 48225 pAC93-pmax-dCas9VP160 : dCas9VP160 on pmax expression vector. 48226 pAC94-pmax-dCas9VP160...Plasmid 48214 pAC1-pCR8-dCas9VP48 : dCas9VP48 on Gateway donor vector pCR8/GW/TOPO. 48218 pAC84-pCR8-dCas9...TOPO. 48219 pAC147-pCR8-dCas9VP64 : dCas9VP64 on Gateway donor vector pCR8/GW/TOPO. 48220 pAC148-pCR8-dCas9VP96...Activators sgRNA Dual Expression ID Plasmid 48236 pAC2-dual-dCas9VP48-sgExpression : Dual expression construct...dCas9VP48 and sgRNA from separate promoters. 48238 pAC152-dual-dCas9VP64-sgExpression : Dual expression construct...dCas9VP64 and sgRNA from separate promoters. 48239 pAC153-dual-dCas9VP96-sgExpression : Dual expression construct...

...want to use double nickase, you could express two spacers and use PX335 to express the Cas9n (nickase). The...PAM sequence to the oligo when cloning my target spacers into the PX330 vector backbone? There is no need...usually adds it when they order the oligo. If your spacer sequence starts with a 'G', you naturally have ... compared to where the target site sequence (protospacer+PAM) is located? If there are other PAMs in the... there needs to be extensive homology of the protospacer followed by PAM. Click here for a target selection... together, with each plasmid having a targeting spacer cloned into the backbone ( pX330 or PX335 , depending...two genomic loci with wildtype Cas9, clone two spacers for each locus into PX330, then co-transfect or...

...Huiwang Ai 73544 pEvol-pAcFRS.2.t1 pAcFRS.2.t1 E. coli p-acetyl-l-phenylalanine (pAcF) Bacterial TAG Farren... Isaacs 73545 pEvol-pAcFRS.1.t1 pAcFRS.1.t1 E. coli p-acetyl-l-phenylalanine (pAcF) Bacterial TAG Farren...141173 pAcBac1-3nitroY-A7-RS A7 3nY tRNA synthetase M. barkeri Mammalian Ryan Mehl 141174 pAcBac1-haloTyrRS...acid Bacerial, Mammalian TAG Peter Schultz 50831 pAcBac2.tR4-OMeYRS/GFP* tyrosyl-tRNA synthetase E. coli... amino acids Mammalian TAG Peter Schultz 50832 pAcBac1.tR4-MbPyl PylRS M. barkeri variety of unnatural...barkeri Tetrazine 3.0 Bacterial TAG Ryan Mehl 174081 pAcBac1-NES-Flag-R2-84-MbRS Tetrazine3.0 NES-Flag-R2-84...barkeri acridone Bacterial TAG Ryan Mehl 174101 pAcBac1.tR4-AcdRS82 Acridone 82 tRNA synthetase M. barkeri...

...referred to as CRISPR ( C lustered R egularly I nterspaced S hort P alindromic R epeat) technologies. Before...for Cas-binding and a user-defined ∼20-nucleotide spacer that defines the genomic target to be modified....target is present immediately adjacent to a P rotospacer A djacent M otif (PAM) The PAM sequence (NGG)... an active, DNA-binding configuration, with the spacer region of the gRNA left free to interact with the...Cas9 will only cleave a given locus if the gRNA spacer sequence shares sufficient homology with the target...throughout the genome, called off-targets, that can impact your experiment. There are many online tools available...Cas9’s interactions with DNA phosphate backbone HypaCas9 - increase Cas9 proofreading and discrimination...