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:
| 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 | 10ng hEF1a:rtTA 10ng TRE:Gal4VP16 10ng UAS:mKate 10ng hEF1a:eYFP 10ng hEF1a:eBFP | 20ng hEF1a:rtTA 20ng TRE:Gal4VP16 20ng UAS:mKate 20ng hEF1a:eYFP 20ng hEF1a:eBFP | 50ng hEF1a:rtTA 50ng TRE:Gal4VP16 50ng UAS:mKate 50ng hEF1a:eYFP 50ng hEF1a:eBFP |
100ng hEF1a:rtTA 100ng TRE:Gal4VP16 100ng UAS:mKate 100ng hEF1a:eYFP 100ng hEF1a:eBFP | 200ng hEF1a:rtTA 200ng TRE:Gal4VP16 200ng UAS:mKate 200ng hEF1a:eYFP 200ng hEF1a:eBFP | ||||
| 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 | 10ng hEF1a:rtTA 10ng TRE:Gal4VP16 10ng UAS:mKate 10ng hEF1a:eYFP 10ng hEF1a:eBFP | 20ng hEF1a:rtTA 20ng TRE:Gal4VP16 20ng UAS:mKate 20ng hEF1a:eYFP 20ng hEF1a:eBFP | 50ng hEF1a:rtTA 50ng TRE:Gal4VP16 50ng UAS:mKate 50ng hEF1a:eYFP 50ng hEF1a:eBFP |
100ng hEF1a:rtTA 100ng TRE:Gal4VP16 100ng UAS:mKate 100ng hEF1a:eYFP 100ng hEF1a:eBFP | 200ng hEF1a:rtTA 200ng TRE:Gal4VP16 200ng UAS:mKate 200ng hEF1a:eYFP 200ng hEF1a:eBFP |
Results: