...Reagents HEK293 cells Recombinant antibody plasmid DNA 500 mL vented flask 50 mL conical tubes Microcentrifuge..., Millipore Sigma A6279 Before Starting Warm the DNA and working stock of PEI-MAX to room temperature ...Transfer the PEI-MAX and recombinant antibody plasmid DNA sample to the biosafety cabinet and incubate for ...cabinet. Add 180 µg of recombinant antibody plasmid DNA to one tube of 6 mL BCD TFX. Cap the tube and vortex...1 to 1:6. Add the diluted PEI-MAX to the diluted DNA. Cap the tube and vortex with three 1 s pulses. Incubate...cells to the biosafety cabinet. Add 12 mL of PEI-MAX/DNA mix to the flask dropwise. Cap the flasks and swirl...Pro-Tip The feed can be repeated up to 4 times for a total of 16% of the culture volume. At 72-96 h post-transfection...

...measure the DNA concentration, use 1 μL) 2 μL 10x NEB T4 DNA ligase buffer 1 μL NEB T4 DNA ligase add ...#B7001S NEB buffer 2 NEB #B7002S T4 DNA ligase NEB #M0202S T4 DNA ligase buffer NEB #B0202S DH5 alpha ...will increase the chance of damaging the DNA. Purify the DNA using a Qiaquick gel extraction kit. Elute...kit to obtain DNA. 3. Conduct a restriction digest with EcoRI and NcoI: 1 μg miniprep DNA 2 μL 10x NEB ...transfect plasmid DNA directly into the target cells. The shRNA will be expressed, but the DNA is unlikely ... pLKO.1 TRC-cloning vector (maxiprep or miniprep DNA) 5 μL 10x NEB buffer 1 1 μL AgeI add ddH 2 O to bring...ddH 2 O Incubate at 37°C for 2 hours. Run digested DNA on 0.8% low melting point agarose gel until you can...

...purified AAV samples with DNase I to eliminate any contaminating plasmid DNA carried over from the production...add carrier DNA to a final concentration of 4 ug/mL to each standard dilution. Dilute DNase-treated and...nuclease free water (uL) Dilution factor Total dilution Dilution 1 (DNase step) 5 uL AAV stock 45 uL 10X 10X...Universal" SYBR master mix which contains a high-quality DNA polymerase and a blend of dTTP/dUTP to minimize carryover...ITR-containing plasmid for standard curve RNase-free DNase 10X DNase buffer Nuclease-free water Microcentrifuge ...production process (DNase does not penetrate the virion). 5 μL sample + 39 μL H 2 O + 5 μL 10X DNase buffer + 1... 1 μL DNase Gently mix sample (do not vortex) Incubate 30 min at 37 °C Transfer to ice ** Critical: do...