.... Pro-Tip Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands...total per 10 cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA 27.8 μg), this...produced, lentivirus can be used for a variety of downstream applications such as stable-cell line generation...T75 flask in 15 mL DMEM Complete. Do not add pen-strep to the media. Use cells that are below passage 15...

...technique is especially useful for introducing promoters, terminators, and other short sequences into the...creating a contiguous DNA fragment. Pro-Tip Add Extreme Thermostable Single-Stranded DNA-Binding protein...

... Pro-Tips Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands...control the multiplicity of infection (MOI) in downstream studies. This protocol was developed for 293T...fluorescent cells, these cells can still be used in downstream applications. Safety Warnings Lentivirus is generally...Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008... after 20–30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and...

.... Pro-Tip Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands...control the multiplicity of infection (MOI) in downstream studies. This protocol was developed for A549...Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008... after 20–30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and...target cell line needs to be empirically determined. Treat the target cells with a range of doses of antibiotic...vector pRosetta . The following day, cells were treated with puromycin. Colonies were allowed to grow in...EF1α dCas9-KRAB . The following day, cells were treated with puromycin. Colonies were allowed to grow in...

...However, the same technique can be used to move promoters, selectable markers, or any other DNA element ...single enzyme, but this will require phosphatase treatment of your recipient plasmid as well as a specifically...re-circularization of the recipient plasmid. You should treat your digested recipient plasmid with a phosphatase...

... °C. Pro-Tip Different brands and FBS lots can promote or inhibit transfection. Test a variety of brands...Microcentrifuge tubes, Neptune 3745.X 6-well tissue culture treated dish, Corning 3516 15 mL conical tubes, VWR 21008... after 20-30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and... reagent for your target cell line. To do this, treat target cells with a range of doses of antibiotic... try higher/lower dilutions depending on your downstream applications. If you’ve titered your virus beforehand...

...10878/ . Description Vector Element U6 Human U6 promoter drives RNA Polymerase III transcription for generation...transduced cells. hPGK Human phosphoglycerate kinase promoter drives expression of puromycin. Puro R Puromycin...with effective gene silencing: Starting at 25nt downstream of the start codon (ATG), search for 21nt sequences...position 15-19. Avoid targeting introns. Avoid stretches of 4 or more nucleotide repeats, especially repeated...Bovine Serum (FBS) Invitrogen: #16000 Penicillin/Streptomycin Invitrogen: #15140-122 Polypropylene tubes VWR...be passaged in DMEM + 10% FBS with penicillin/streptomycin, cells should be plated at this step in DMEM...10% FBS without antibiotics (no penicillin or streptomycin). Day 2: b. Perform the transfection in the ...