...
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:
...
...
...
...
...
Results:
| Expand | ||
|---|---|---|
| ||
Adherent Transfection
| Untransfected | 10ng | 25ng | 50ng | 100ng | 250ng | 500ng | 1000ng | |
| 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:
...
| title | Plate map |
|---|
...
...
...
100ng TRE: |
...
Gal4VP16 100ng UAS: |
...
eYFP 100ng |
...
100ng |
...
100ng 1000 nm Dox |
...
200ng TRE: |
...
Gal4VP16 200ng UAS: |
...
eYFP 200ng |
...
200ng |
...
200ng
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
Red vs. Blue | ||||||||
Yellow vs. Blue | ||||||||
Co-Transfection Efficiency | 0% | 0% | 0.053% | 0.12% | 0.74% | 4.33% | 5.37% | 3.15% |
Red vs. Blue | ||||||||
Yellow vs. Blue | ||||||||
Co-Transfection Efficiency | 0% | 0% | 0.047% | 0.29% | 1.23% | 5.87% | 5.62% | 2.91% |
Co-Transfection Efficiency Average | 0% | 0% | 0.05% | 0.205% | 0.985% | 5.1% | 5.945% | 3.03% |
Suspended Transfection
| Untransfected | 10ng | 25ng | 50ng | 100ng | 250ng | 500ng | 1000ng |
...
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 |
...
Co-Transfection Efficiency 1 | 0% | 0% | 0.063% | 0.19% | 1.07% | 4.61% | 3.95% | 2.3% |
Co-Transfection Efficiency 2 | 0% | 0% | 0.066% | 0.32% | 0.94% | 4.3% | 4.77% | 2.23% |
Co-Transfection Efficiency Average | 0% | 0% | 0.0645% | 0.255% | 1.005% | 4.455% | 4.36% | 2.265% |
*performed a T-test on data (albeit sketchy T-test, given that there were only 2 duplicates of each condition) --> suspended vs. adherent show no significant difference in co transfection efficiency |
Discussion:
Bleed-through:
We should run single color controls to account for bleed-through. The gating that gave the above data excluded a population when gating in the yellow vs blue space on account of bleed through. However, we can't know for sure that that population is just bleed through without single color controls.
Non-linear plot:
 | y is just y=t x=0.1t+2 So early on the +2 dominates, but at the higher domain the 0.1t dominates. That is why the plot looks like it does. All a*x vs b*y linear functions are straight diagonal lines. ax+c looks like ax for ax>>c, and like c for ax<<c. and a constant makes a vertical lines on log log axis. That is why it starts pointing up then tilts over and makes a straight diagonal. |
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 |
...
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
...