August 11th, 2011
- Checked transformation plates. Success with Sigma-Aldrich reagents and a little bit of growth with Kozminski’s reagents. Kozminski said that perhaps the Sigma-Aldrich reagents are newer, so it allowed for more efficient transformation. He also said that before we test our selected cells, we should perform a second selection on another dropout plate so that we make sure our cells are definitely transformed an not cells that may be “teetering on the edge of life.” Another suggestion was that we make a stock from our cells transformed with the empty vector so that we have a control to compare our experiments and make sure the cells are growing correctly.
- PCR purified products from the day before and ran on a gel:
5. PCA 2
6. VEGF amplified
- We found that the 1-12 reaction ran very well because we saw a strong band at 500bp, but something went wrong with the 13-22 pca reaction. This resulted in no yield for the final construct pca reaction as well as the pcr amplification. We decided to re-run the 13-22 pca reaction in 3 groups: 13-22, 13-58, 59-62. Then, we visualized the products on a gel:
- We obtained nothing because we didn’t realize the gel didn’t run and let it sit in TAE buffer for an hour then ran it for another hour. Thus, we rerun samples on a fresh gel:
- The results showed that we got nothing so a picture was not taken.
- Side note: we found out how to submit the backbone and the vegf together, but still maintain that they are two separate novel parts.