Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/04/28

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Overview

The KAH187-MV8 plasmid is a mammalian expression vector containing the CMV promoter, followed by two copies of AmCyan with a nuclear localization signal. We'll be using it as a control for transfection studies, but the Neon transfection unit asks for plasmid at a concentration of at least 1,000 ng/µL. I earlier performed a plasmid maxiprep and collected >1 mL of KAH187-MV8 at 164 ng/µL. Today I plan to concentrate 10x, for a final concentration of >1,000 ng/µL in 100 µL final volume.

DNA Concentration

This is a classic nucleic acid precipitation/resuspension protocol. Instructions taken from the Sigma GenElute Plasmid Maxiprep protocol.

  1. Add 0.1 volumes of 3.0M sodium acetate buffer solution at pH 5.2 and 0.7 volumes of isopropanol to the recovered plasmid. Mix well by inversion and centrifuge at >15,000g for 30 minutes at 4°C.
  2. Decant supernatant, being careful not to disturb the pellet. Rinse the pellet with 1.5 mL 70% ethanol and centrifuge at >15,000g for 10 minutes at 4°C.
  3. Decant supernatant, being careful not to disturb the pellet. ALlow pellet to air-dry until residual ethanol has evaporated.
  4. Resuspend the pellet in the desired volume of mol. bio. grade H2O (100 µL in this case).

Results

Sample ID Conc (ng/µL) St Dev (ng/µL) 260/280
KAH187-MV8 original 173.1 0.9001 1.89
KAH187-MV8 10x conc 1518 29.16 1.87

Success! With an 88% yield, no less. There's some of the original plasmid left over as well. Both samples stored at -20°C in my 2nd box.