IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-8

Analysis of yesterday's {KaiA/B + bkb}\X-P digest

All the lanes have a band around 3400 bp, which is the length of an uncut J04450 device (RFP insert + bkb). We think that our backbone extraction from 2006-8-2 was contaminated with uncut J04450, which would dominate the subsequent ligations. Thus, we will rerun the 2006-8-2 digest for a longer time, and run the gel longer as well before extracting.

One piece of evidence against the above hypothesis is the observation that our KaiA + bkb and KaiB + bkb ligations do not fluoresce red, as one would expect if they had been transformed with uncut J04450.

Lanes 3, 5, and 8 appear to contain the KaiA insert (which should be 903 bp). However, we're not sure why 1) There's still a 3400 bp band in those lanes, and 2) There's no backbone (2000 bp) in those lanes. It's possible that these cultures were inoculated from several colonies, but we were careful about choosing single colonies.

Todo

(in terms of priority)

• Talk about Western blotting

• Digest lane 3 (10uL DNA) like done yesterday, and run it against undigested sample (10uL DNA). Likewise for lane 8 and lane 12 and lane 16. Let the digest go for 6 h; use more enzyme. (XP)

• J04550 RFP+backbone needs to be likewise digested (ES) and run against undigested form. Carefully gel purify out the 2079b backbone. CIP, re-ligate, etc... (tomorrow most likely)

• Religate and transform low copy plasmid backbone with RFP (Jeff?)

• Analyze sequencing results (Peng)

• Miniprep more of lane 3, 8, 12, and 16 from yesterday's digest.

• Perry's experiments