...μg pLKO.1 shRNA plasmid 750 ng psPAX2 packaging plasmid 250 ng pMD2.G envelope plasmid to 20 μl serum-...to vary the ratio of shRNA plasmid, packaging plasmid, and envelope plasmid to obtain the ratio that gives...Producing Lentiviral Particles E.1 Recommended materials E.2 Protocol for producing lentiviral particles...shRNA. psPAX2 Packaging plasmid for producing viral particles. pMD2.G Envelope plasmid for producing viral... also be used with packaging plasmid pCMV-dR8.2 dvpr and envelope plasmid pCMV-VSVG from Robert Weinberg...Due to the long terminal repeats found in lentiviral plasmids, Addgene currently recommends using a strain....1 Protocol pLKO.1 - TRC Cloning Vector Addgene Plasmid 10878. Protocol Version 1.0. December 2006. Copyright...

... 0.72 pmol Transfer Plasmid* 1.64 pmol OptiPro SFM to total volume 500 μL *Plasmid concentrations and ... each transfer plasmid. Pro-Tip Endotoxins can inhibit transfection; therefore, plasmid DNA purification...endotoxin removal step. For high quality plasmid DNA, the plasmids should also be propagated in an endonuclease... cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA 27.8 μg), this would be...This protocol can be used to produce lentivirus from a lentiviral vector transfected into 293T cells using...~20 h. Prepare a mixture of the 3 transfection plasmids into OptiPro SFM: Reagent Amount per 10 cm dish...is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 ...

...cells that have taken up the lentiviral transgene. Note that not all lentiviral vectors deliver antibiotic...Thaw the lentiviral aliquot on ice prior to use. Prepare a range of dilutions of the lentivirus in DMEM.... This is because lentiviral integration is random. Given the MOI of the lentivirus used, cells may also...an integrated lentiviral vector. Protocols...expressing a gene of interest from an integrated lentiviral vector. Unlike the short-term protein expression...transfection approaches, generating cell lines using lentiviral vectors enables long-term protein expression ...associated with repeated transient transfection. Some lentiviral vectors deliver mammalian antibiotic resistance...

...ddPCR Lentivirus sample data, RPP30 positive (green) and negative (black) droplets Sample ID Plasmid ID ...be close to zero (A01). In this protocol, the lentiviral particles are serially diluted and used to transduce...Y, Bergelson S, Feschenko M. Determination of Lentiviral Infectious Titer by a Novel Droplet Digital PCR... of Lentivirus Vectors You may also like... Lentivirus Colony Formation Titration Assay Lentivirus Fluorescence... PCR (ddPCR) for lentivirus vector titration. Protocols... Protocols ddPCR Titration of Lentivirus Vectors ddPCR Titration...Assay AAV Titration by qPCR Addgene Viral Service Lentivirus Guide Virus Blog Posts Introduction This protocol...

... here ). Lentiviral plasmids can recombine between their LTRs resulting in a smaller plasmid containing...differences, and plasmid recombination can all impact the representation of individual plasmids in the pooled...protocol to perform amplification of CRISPR pooled plasmid libraries in Escherichia coli cells. Protocols... protocol allows the amplification of a pooled-plasmid library in Escherichia coli cells. Pooled libraries...libraries contain tens to millions of different plasmids in a single sample. They are often used for screening...throughput multiplexed experiments. These pooled plasmids differ by only a short region of DNA: guide RNAs...results for libraries up to 200,000 individual plasmids and can be scaled according to the actual number...

...complicated procedures. Plasmid Cloning Protocols for constructing and analyzing your plasmids. Virus Protocols...Amplify CRISPR pooled-plasmid libraries Diagnostic Restriction Digest Verify a plasmid using restriction ...Transfection Introduce plasmid DNA to mammalian cells Lentivirus Production Produce lentivirus with a polyethyenimine... that can be used for basic molecular biology, plasmid cloning, and titering and testing your viral preparations...Bacterial Glycerol Stocks Store bacterial strains or plasmids for long term use Watch the Video! DNA Purification...spectrophotometer Restriction Digests Cut and analyze plasmid DNA with restriction enzymes Agarose Gel Electrophoresis... the Video! Sequence Analysis Verify important plasmid features using sequence analysis RNA Extraction...

...lentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid #52962 ) and is described in Improved vectors and... pool of stable cells. Transducing cells with lentivirus results in a heterogenous polyclonal population...protocol for generating stable cell lines with lentivirus . Day 0: (optional) Seed cells for the generation...protocol for generating stable cell lines with lentivirus . Pro-Tip Because the polyclonal cell pool is...