AIM/HYPOTHESIS
Due to an error in the previous protocol, the amount of DNA used for transfections in 1. Suspension culture and MT302 + LD401 transfection of HEK293TLD cells was only 1/20th of the required amount. To test whether this was in fact the issue, I performed a small scale transfection with MT302. To also check for loss of efficiency after passing the cells post transfection I took along one flask that was split, regrown and then induced.
MATERIALS
See 1. Suspension culture and MT302 + LD401 transfection of HEK293TLD cells
METHODS
Cells were cultured in 125mL baffled suspension culture flasks under 8% CO2, 80% humidity @ 37°C. Grown up to 3M cells/mL in two flasks. Transfection was done with 50µg of DNA and 150µg of PEI Max in 2.5mL of hybridoma SFM in both flasks. One flask was induced with 1µg/mL dox 24 hours later and harvested another 24 hours later. The other flask was split 1:4 24 hours post transfection, induced 48 later and harvested 24 hours post induction.
Samples:
1) Regular; 750mg WCW
2) 1:4; 680mg WCW (x4)Gel was loaded as follows:
1) Marker
2) 1 – 10µg
3) 1 – 20µg
4) 1 – 50µg
5) [space]
6) 2 – 10µg
7) 2 – 20µg
8) 2 – 50µg
9) [space]
10) MT289 – 7.5µg
11) MT289 – 15µg
12) Marker Lower panel of the blot was stained with a-ORF1, top panel with a-Flag and after stripping with a-ORF2. RESULTS
ORF1
ORF2
DISCUSSION