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Progress:

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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:

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CloningTransfectionDoxCytometryData Analysis
waiting on TRE:Gal4VP16    

 

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 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

    

...

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 

Adherent Seeding

Adherent & Suspended Transfections

Addition of Doxycycline

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Results:

 

Discussion: