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

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Owwnotebook icon.pngTest transfection of U2OS using pMaxGFP, KAH187-MV8, and KAH126-MV2 Report.pngMain project page
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Overview

Transfection efficiency has been much lower than anticipated despite numerous attempts to optimize. Here I'm going to try transfection with three different plasmids: pMaxGFP (positive control from Lonza), KAH187-MV8 (expresses CFP using CMV promoter), and KAH126-MV2 (expresses PcTF-RFP using CMV promoter). Will see how they differ in effectiveness.

Each treatment will use be performed in duplicate on a 6-well plate.

Transfection Setup

See the Haynes Lab transfection protocol for details.

One difference: final volume of antibiotic-free medium in each well is 2mL, not 4mL. All other values kept the same.

Plasmid Conc (ng/µL) Vol DNA (µL) Vol water (µL)
pMaxGFP 348 5.7 14.3
KAH187-MV8 1518 1.3 18.7
KAH126-MV2 809 2.5 17.5
Reagent Volume (µL)
DNA 20
Opti-MEM 570
PLUS reagent 2.5
Lipofectamine LTX 7.5