Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/05/20

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I'm making a lot of the pMaxGFP plasmid for use in transfection (Neon transfection, lipofectamine, etc.), as these processes require a very high concentration of very pure DNA.


Yesterday I set up a 150mL culture of LB-Ampicillin from a DH5α-Turbo colony transformed with the pMaxGFP plasmid. I spun down the culture at 5000g for 10 minutes and discarded the supernatant, then followed the instructions for plasmid prep as laid out in the Sigma GenElute HP Endotoxin-Free Plasmid Maxiprep Kit.

Plasmid concentration was measured on the Take3 Nanodrop.

DNA Quantification

Final volume of the purified plasmid is approximately 1.5 mL.

Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280
pMaxGFP 486.333 7.966264997 1.888

I took 100µL of the purified plasmid and transferred it to a new sterile tube and am leaving that tube open in the BSC overnight to evaporate off and concentrate. Tomorrow I'll also perform a sodium acetate-ethanol precipitation with another aliquot of the plasmid.