...cells several days later, and isolating genomic DNA. This DNA is then used as a template to amplify a portion...of the viral genome and the absence of unexpected DNA contaminants. The data is also used to determine ...abundance of viral capsid proteins as a fraction of total protein present in the sample is also determined...proteins VP1, VP2, and VP3 are shown relative to the total protein present in the sample. Sterility Viral vector...lentiviral vector, incubated for 72 h, and genomic DNA is extracted. Primers and probes targeting integrated...matching system that follows the sample from the DNA tube all the way to the cleared viral preparation...

...pellets in 50 μl of DNA extraction solution. NOTE: Generally ~100,000 cells are used for DNA extraction, although...recommend an efficient method for DNA extraction, any method for genomic DNA isolation may be utilized to ...for a total volume of 200 μl. Wait 24 - 72 hr to allow cells to grow. Aspirate media. Add 50 μl DNA extraction...evaluate clones at the DNA as well as RNA and/or protein levels. To evaluate the DNA, amplify deletion bands...sufficient cell proliferation for screening genomic DNA (gDNA) for the intended deletion by PCR (see steps 6.1...C for 6 min, 98 °C for 2 min to extract gDNA. Measure the DNA concentration. NOTE: While steps 6.1 and...DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair...

...viral vector preps, which includes MTA facilitation, DNA amplification, and high-quality quality control measures...actual yields may vary by construct. Size Quantity Total Volume Minimum Titer Typical Titer Small 10 × 100...including legal approval and an implementation letter DNA amplification Viral vector production Density centrifugation...Quality is our priority. During this time, we amplify DNA, produce the prep, and perform comprehensive quality...

...better for PCR and surveyor. I used DNA polymerase Takara Ex Taq ™ DNA Polymerase for my genomic PCR, but... single stranded DNA (ssDNA) oligo which usually work better than plasmids. For ssDNA oligo design, the...200ng to 500ng of 293FT genomic DNA as template after performing gDNA purification and have tried conditions...the Zhang lab typically uses around 100-150bp total homology. The mutation is introduced in the middle...seem much shorter. What is the maximum amount of DNA that can be inserted into the genome using CRISPR...solution to perform this step to extract genomic DNA from cells. The Zhang lab uses around 50ul of the...small (<50bp) changes, you should consider using ssDNA oligo as donor template, it's usually more efficient...

...PI Lentiviral Generation gRNAs per gene Total gRNAs Adamson DNA Repair CRISPRi Libraries 177663, 177664...Varies CRISPR/Cas9-assisted Removal of Mitochondrial DNA (CARM) Library 82480 Knockout Mouse Xie N/A N/A 395...Inhibition Mouse Weissman 3rd 5 10 107,415 214,830 DNA Damage Response MKOv4 Library 140219 Knockout Human...110066 Knockout Human Sabatini 3rd 10 30,290 Human DNA Binding Domain-Focused CRISPR Knockout Library 123334...

...interactions between DNA and protein. Micrococcal nuclease digestion is used to shear the DNA in these assays... uses antibodies to capture protein:DNA complexes. After capture, the DNA is released from the complex...suitable for DNA:histone modifier interactions, which have strong binding between the protein and DNA. Some ...Alternatively, the samples can be normalized to a total protein measurement. Both methods will also serve...light. Shear the samples using sonication to break DNA up into fragments of 300-1000 bps in length. Note...IP section above. Reverse the crosslink, isolate DNA, and perform downstream analysis. Special considerations...gentler but results in non-random fragmentation of the DNA. Controls for capture methods Capture methods should...