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Procedure:

The plate map is as follows. Using lipo3k protocol. There will be two plates using this platemap, one with suspended transfection and one with adherent transfection.

HEK293

hEF1a:rtTA 2ng

TRE:Gal4VP16 2ng

UAS:eYFP 2ng

hEF1a:mKate 2ng

hEF1a:eBFP 2ng

 

1000 nm Dox

hEF1a:rtTA 5ng

TRE:Gal4VP16 5ng

UAS:eYFP 5ng

hEF1a:mKate 5ng

hEF1a:eBFP 5ng

 

1000 nm Dox

hEF1a:rtTA 10ng

TRE:Gal4VP16 10ng

UAS:eYFP 10ng

hEF1a:mKate 10ng

hEF1a:eBFP 10ng


1000 nm Dox

hEF1a:rtTA 20ng

TRE:Gal4VP16 20ng

UAS:eYFP 20ng

hEF1a:mKate20ng

hEF1a:eBFP 20ng


1000 nm Dox

hEF1a:rtTA 50ng

TRE:Gal4VP16 50ng

UAS:eYFP 50ng

hEF1a:mKate 50ng

hEF1a:eBFP 50ng


1000 nm Dox

hEF1a:rtTA 100ng

TRE:Gal4VP16 100ng

UAS:eYFP 100ng

hEF1a:mKate 100ng

hEF1a:eBFP 100ng


1000 nm Dox

hEF1a:rtTA 200ng

TRE:Gal4VP16 200ng

UAS:eYFP 200ng

hEF1a:mKate 200ng

hEF1a:eBFP 200ng

 

1000 nm Dox

    
HEK293

hEF1a:rtTA 2ng

TRE:Gal4VP16 2ng

UAS:eYFP 2ng

hEF1a:mKate 2ng

hEF1a:eBFP 2ng

 

1000 nm Dox

hEF1a:rtTA 5ng

TRE:Gal4VP16 5ng

UAS:eYFP 5ng

hEF1a:mKate 5ng

hEF1a:eBFP 5ng

 

1000 nm Dox

hEF1a:rtTA 10ng

TRE:Gal4VP16 10ng

UAS:eYFP 10ng

hEF1a:mKate 10ng

hEF1a:eBFP 10ng

 

1000 nm Dox

hEF1a:rtTA 20ng

TRE:Gal4VP16 20ng

UAS:eYFP 20ng

hEF1a:mKate20ng

hEF1a:eBFP 20ng

 

1000 nm Dox

hEF1a:rtTA 50ng

TRE:Gal4VP16 50ng

UAS:eYFP 50ng

hEF1a:mKate 50ng

hEF1a:eBFP 50ng

 

1000 nm Dox

hEF1a:rtTA 100ng

TRE:Gal4VP16 100ng

UAS:eYFP 100ng

hEF1a:mKate 100ng

hEF1a:eBFP 100ng


1000 nm Dox

hEF1a:rtTA 200ng

TRE:Gal4VP16 200ng

UAS:eYFP 200ng

hEF1a:mKate 200ng

hEF1a:eBFP 200ng

 

1000 nm Dox

    

Progress: 

CloningTransfectionDoxCytometryData Analysis

 

    

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.

Approach:

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.

Parts Needed:

PartStatus
hEF1a:rtTa 
TRE:Gal4VP16 
UAS:mKate 
hEF1a:eYFP 
hEF1a:eBFP 

 

Results:

 

Discussion:

 

 

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