Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/04/08

RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines

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Verification of KAH187-MV8 plasmid When troubleshooting the transfection of K562 cells, we need to use a positive control with the same promoter and backbone as our PcTF plasmid to determine the cause of low transfection efficiency - ie, is the uptake of plasmid low, or are cells not transcribing PcTF-RFP when they're transfected? To that end I'm making more of the KAH187-MV8 plasmid, which contains a CMV promoter with two copies of the AmCyan gene and a nuclear localization sequence. Ran a plasmid miniprep on two cultures selected from transformation of DH5α-Turbo competent E. coli. DNA concentration: Sample ID ng/µL avg ng/µL stdev KAH187-MV8 colony 1 635.6855 16.37588595 KAH187-MV8 colony 2 350.219 4.553767671 Digestion parameters: Reagent Volume (µL) FD 10x Buffer 2 water 15 DNA (1µg) 1 to 3 EcoRI 1 ApaLI 1 Gel image: Expected size fragments (from Benchling): 3941, 1243, 1135, 288, 234 I'm not sure what's going on with this digestion. Colony 2 appears to match up with the expected fragments, except there's an extra band below the 3941 fragment. Colony 1's bands appear to be all over the place.