Haynes Lab:Notebook/RNA-seq of PcTF Transfected U2OS & SK-N-SH cell lines/2015/05/19

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Owwnotebook icon.pngSetup for K562 transfection optimization Report.pngMain project page
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Overview

K562 cells are in PS-α right now. Once they're ready for expansion into PS-β, I'll be setting aside a fraction to grow in antibiotic-free medium for a transfection optimization experiment. Here's the general layout of the transfection procedure I'll be performing.

Screenshot 2015-05-19 10.42.49.png

Procedure

1. Prepare master mixes of Opti-MEM, DNA, & PLUS Reagent.

Sample ID Opti-MEM (µL) DNA (µg) PLUS (µL)
1 50 4 5
2 50 1 5
3 50 0.4 5
4 50 6 5
5 50 1.5 5
6 50 0.6 5
7 50 8 5
8 50 2 5
9 50 0.8 5
10 50 10 5
11 50 2.5 5
12 50 1.0 5

2. Prepare master mixes of Opti-MEM and LTX Reagent.

Sample ID Opti-MEM (µL) LTX (µL)
1 50 2
2 50 2
3 50 2
4 50 3
5 50 3
6 50 3
7 50 4
8 50 4
9 50 4
10 50 5
11 50 5
12 50 5

3. Incubate at room temperature for 5 minutes, then combine the two master mixes for each sample (100+µL total for each sample).

4. Take cells out of the incubator and add 50 µL of mixed DNA + LTX into each well (duplicates for each sample).