Plan+scFv+ligation+10-3-12

10-3-12

I discovered that although the SfiI site was removed, the appropriate NotI site was added, and the appropriate NotI site was removed in the pComb3XSS v2 vector, there was some extra sequence near the removed NotI site. I don't think this should adversely affect anything though.

Plans sequence original pcomb and modified pcomb with new primers to check right in-fusion site clone modified notI PCR fragment into pJET Cut pcomb3xssv2 with sfiI and NotI and add stuffer for test

The main thing I need to do is proceed with the plasmid, and cut with sfiI and NotI. I'd like to do a practice fragment addition (rather than ligating in the actual scFv genes). I'll use rCompTT for the fragment. The experiment on 12-7-10 has more info about about this digestion. see pCMVils-rCompTT

10-4-12 Started proceeding with test ligation with a cut with SfiI. Placed sample at -20 C 10-5-12 Removed sample from -20 C. Performed NotI digest. Ran gel for gel extraction.

Expected rCompTT fragment after digesting with NotI and SfiI: 1.3 kb Expected pComb fragment after digesting with NotI and SfiI: 1.677 kb