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

Second batch (B2): We inoculated some toxT gRNA. Today we are minipreping it. (It already passed the test digest.

Third batch (B3): Yesterday, we inoculated 2 reporter-Chlora colonies from each plates. Today we digest them, gel purify, ligate, and transform them onto Carb back bone, along with re-ligating and re-transforming ctxA that kept failing. Gel test for some were not good so we inoculated a few more (from tcpR).

Forth batch (B4): We transformed B4 reporters on promoter-Chlora. The plates are good today. There's one colonies on the negative and the colonies on other plates appear with different sizes, so we are picking 2 colonies from each plates to inoculate.

We remade some gRNA from the primers. Unfortunately, yesterday we didn't have the machine for overnight ligation. So we will ligate it tonight.

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