Procedure:
Cloning| Expand |
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TRE:Gal4VP16 LRed
Procedure:
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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.
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5ng hEF1a:eYFP
5ng hEF1a:eBFP
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Results:
Adhered Transfection:
Discussion:
Progress:
| Cloning | Transfection | Dox | Cytometry | Data Analysis |
|---|---|---|---|---|
| 07/06 | 07/07 | 07/08 |
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 |
| 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 |
Procedure:
| Expand | ||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ||||||||||||||||||||||||
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.
|
Results:
Adhered Transfection:
Discussion:
Progress:
| Cloning | Transfection | Dox | Cytometry | Data Analysis |
|---|---|---|---|---|
| 07/06 | 07/07 | 07/08 |
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
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:
| Part | Status |
|---|---|
| hEF1a:rtTa | |
| TRE:Gal4VP16 | |
| UAS:mKate | |
| hEF1a:eYFP | |
| hEF1a:eBFP |
| 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 |
Results:
Discussion: