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 B3, we need to re-do whatever we did today because of new inoculation of the old plates. Meaningly, miniprep, digest, gel purify, ligate, and transform newly inoculated tcpR. we should have colonies of B3 reporters on the plates we are transforming today. We should miniprep the B4 reporters we inoculated today. For gRNA of B4, ligation will be done and we will transform them tomorrow. Saturday, we will inoculate. 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