Transfection DNA Mass Optimization

Background:

Because transfecting a large number of plasmids (~8) into HEK293 cells can drastically increase cytotoxicity and lower transfection efficiency, we are optimizing our transfections before we start characterizing the B-Cell Receptor. We plan on evaluating suspended vs. adherent transfection and varying total mass of DNA transfected. Because we will be transfecting a large protein complex (BCR) into our cells we want to use many different plasmids interacting to test our transfection efficiency.

Parts Needed:

hEF1a:rtTA

TRE:Gal4VP16

UAS:mKate

hEF1a:eYFP

hEF1a:eBFP

Procedure:

We will be testing in duplicate 10, 25, 50,100, 250, 500, and 1000ng of DNA with lipo 3K suspended vs non suspended transfection to determine optimal transfection conditions for our cells.

The plate map is as follows. Using lipo3k protocol.

HEK293 Plain

2ng hEF1a:rtTA

2ng TRE:Gal4VP16

2ng UAS:mKate

2ng hEF1a:eYFP

2ng hEF1a:eBFP

5ng hEF1a:rtTA

5ng TRE:Gal4VP16

5ng UAS:mKate

5ng hEF1a:eYFP

5ng hEF1a:eBFP