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Progress:
| Cloning | Transfection | Dox | Cytometry | Data Analysis |
|---|---|---|---|---|
TRE:Gal4VP16 LRed |
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:mKate 2ng hEF1a:eYFP 2ng hEF1a:eBFP 2ng
1000 nm Dox | hEF1a:rtTA 5ng TRE:Gal4VP16 5ng UAS:mKate 5ng hEF1a:eYFP 5ng hEF1a:eBFP 5ng
1000 nm Dox | hEF1a:rtTA 10ng TRE:Gal4VP16 10ng UAS:mKate 10ng hEF1a:eYFP 10ng hEF1a:eBFP 10ng 1000 nm Dox | hEF1a:rtTA 20ng TRE:Gal4VP16 20ng UAS:mKate 20ng hEF1a:eYFP 20ng hEF1a:eBFP 20ng 1000 nm Dox | hEF1a:rtTA 50ng TRE:Gal4VP16 50ng UAS:mKate 50ng hEF1a:eYFP 50ng hEF1a:eBFP 50ng 1000 nm Dox |
hEF1a:rtTA 100ng TRE:Gal4VP16 100ng UAS:mKate 100ng hEF1a:eYFP 100ng hEF1a:eBFP 100ng 1000 nm Dox | hEF1a:rtTA 200ng TRE:Gal4VP16 200ng UAS:mKate 200ng hEF1a:eYFP 200ng hEF1a:eBFP 200ng
1000 nm Dox | ||||
| HEK293 | hEF1a:rtTA 2ng TRE:Gal4VP16 2ng UAS:mKate 2ng hEF1a:eYFP 2ng hEF1a:eBFP 2ng
1000 nm Dox | hEF1a:rtTA 5ng TRE:Gal4VP16 5ng UAS:mKate 5ng hEF1a:eYFP 5ng hEF1a:eBFP 5ng
1000 nm Dox | hEF1a:rtTA 10ng TRE:Gal4VP16 10ng UAS:mKate 10ng hEF1a:eYFP 10ng hEF1a:eBFP 10ng
1000 nm Dox | hEF1a:rtTA 20ng TRE:Gal4VP16 20ng UAS:mKate 20ng hEF1a:eYFP 20ng hEF1a:eBFP 20ng
1000 nm Dox | hEF1a:rtTA 50ng TRE:Gal4VP16 50ng UAS:mKate 50ng hEF1a:eYFP 50ng hEF1a:eBFP 50ng
1000 nm Dox |
hEF1a:rtTA 100ng TRE:Gal4VP16 100ng UAS:mKate 100ng hEF1a:eYFP 100ng hEF1a:eBFP 100ng 1000 nm Dox | hEF1a:rtTA 200ng TRE:Gal4VP16 200ng UAS:mKate 200ng hEF1a:eYFP 200ng hEF1a:eBFP 200ng
1000 nm Dox |
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:
| Part | Status |
|---|---|
| hEF1a:rtTa | |
| TRE:Gal4VP16 | |
| UAS:mKate | |
| hEF1a:eYFP | |
| hEF1a:eBFP |
Results:
Discussion:
Progress:
| Cloning | Transfection | Dox | Cytometry | Data Analysis |
|---|---|---|---|---|
TRE:Gal4VP16 LRed |
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:mKate 2ng hEF1a:eYFP
2ng hEF1a:eBFP 1000 nm Dox | 5ng hEF1a:rtTA 5ng TRE:Gal4VP16 5ng UAS:mKate 5ng hEF1a:eYFP 5ng hEF1a:eBFP 1000 nm Dox | 10ng hEF1a:rtTA 10ng TRE:Gal4VP16 10ng UAS:mKate 10ng hEF1a:eYFP 10ng hEF1a:eBFP 1000 nm Dox | 20ng hEF1a:rtTA 20ng TRE:Gal4VP16 20ng UAS:mKate 20ng hEF1a:eYFP 20ng hEF1a:eBFP 1000 nm Dox | 50ng hEF1a:rtTA 50ng TRE:Gal4VP16 50ng UAS:mKate 50ng hEF1a:eYFP 50ng hEF1a:eBFP 1000 nm Dox |
100ng hEF1a:rtTA 100ng TRE:Gal4VP16 100ng UAS:mKate 100ng hEF1a:eYFP 100ng hEF1a:eBFP 1000 nm Dox | 200ng hEF1a:rtTA 200ng TRE:Gal4VP16 200ng UAS:mKate 200ng hEF1a:eYFP 200ng hEF1a:eBFP 1000 nm Dox | ||||
| HEK293 Plain | 2ng hEF1a:rtTA 2ng TRE:Gal4VP16 2ng UAS:mKate 2ng hEF1a:eYFP 2ng hEF1a:eBFP 1000 nm Dox | 5ng hEF1a:rtTA 5ng TRE:Gal4VP16 5ng UAS:mKate 5ng hEF1a:eYFP 5ng hEF1a:eBFP 1000 nm Dox | 10ng hEF1a:rtTA 10ng TRE:Gal4VP16 10ng UAS:mKate 10ng hEF1a:eYFP 10ng hEF1a:eBFP 1000 nm Dox | 20ng hEF1a:rtTA 20ng TRE:Gal4VP16 20ng UAS:mKate 20ng hEF1a:eYFP 20ng hEF1a:eBFP 1000 nm Dox | 50ng hEF1a:rtTA 50ng TRE:Gal4VP16 50ng UAS:mKate 50ng hEF1a:eYFP 50ng hEF1a:eBFP 1000 nm Dox |
100ng hEF1a:rtTA 100ng TRE:Gal4VP16 100ng UAS:mKate 100ng hEF1a:eYFP 100ng hEF1a:eBFP 1000 nm Dox | 200ng hEF1a:rtTA 200ng TRE:Gal4VP16 200ng UAS:mKate 200ng hEF1a:eYFP 200ng hEF1a:eBFP 1000 nm Dox |
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:
| Part | Status |
|---|---|
| hEF1a:rtTa | |
| TRE:Gal4VP16 | |
| UAS:mKate | |
| hEF1a:eYFP | |
| hEF1a:eBFP |
Results:
Discussion:
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