Transient Transfection DNA Preparation

DNA for transient transfection was prepared by Miraprep of E. coli DH5α cells as previously described using a Qiagen (Valencia, CA) DNA miniprep kit (Pronobis et al., 2016 (link)). Briefly, transformed E. coli DH5α cells were grown in 50 mL LB media supplemented with ampicillin (50 µg/mL) overnight at 37°C. Cells were collected by centrifugation and resuspended in P1 buffer supplemented with fresh RNase. After alkaline lysis and neutralization, the supernatant was cleared by centrifugation. The supernatant was diluted with an equal volume of 96% (v/v) ethanol prior to loading onto five Qiagen miniprep spin columns. At this point, the DNA was washed and eluted according to the Qiagen protocol. Purity of the eluted DNA was checked by measuring the A_{280 nm}/A_{260 nm} ratio on a NanoDrop 2000c spectrophotometer and by agarose gel electrophoresis. The correct gene sequence for p300 was also confirmed by sequencing prior to transfection in human cells.