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

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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: 2015-04-08 KAH187-MV8 digest annotated.png

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.