Today is also long day. (We almost repeated everything we did yesterday: inoculation, miniprep, ligation, and transformation. Also, sequencing results are here.

Second batch (B2):

• toxT: got very high noise in the background. We are possibly re-sequencing them.

• tcpA: (gRNA+Promoter) first clone was wrong after sequencing. We have two clones so we will re-test-digesting-2%-gel them and send them out for sequencing.

Third batch (B3): Yesterday, we transformed reporter-Carb; today, we inoculate them. The colonies are not very big, we have about 3 colonies on the negative; however, the other plates are very good: they has at least twice-fold the colonies on the negative.

Forth batch (B4): We inoculated yesterday. We are minipreping, digesting, ligating onto Carb back bone, and transforming them

tcpQ is being re-digested and re-tested on 2% gel to make sure of our clone. We tested it on 1% gel but it created a smear and was dubious.

Ran out of PstI-HF!!! Need to order!!!

Tomorrow: For B4 reporters and B3 gRNA, inoculation. B3 reporters: miniprep. Sunday, we will miniprep. Next week, on Monday, we should have everything ready for test digest and then finally send the last batch of sequencing!

• B3 reporter vectors on Chlora: Glycerol Stock + Miniprep + Digest + Gel extract and purify + ligate + transform
• Inoculate 2 more colonies from tcpR plates.
• B4 reporter vectors on Chlora: Inoculate 2 colonies from each plate acfA, acfB, tcpT, tcpQ
• ctxA: ligate + transform
• Miniprep toxT gRNA.
• Overnight ligation of primers gRNA under promoter: acfA, acfB, tcpF