Today is a medium day. Yesterday, we inoculated the rest of the colonies on the plates. Also inoculated third batch of reporter vectors on Carb. So today we have tons (14) to miniprep and test digest. Yesterday transformation of third batch reporter vectors with promoter-Chlora was good: many colonies but about 3 colonies appeared on the negative. We inoculated 2 colonies from each plates.

Fourth batch (B4): We are ligating and transforming the fourth batch reporters on promoter-Chlora. We ran out of toxT gRNA so we are inoculating some more. We are remaking some gRNA from the primers.

Tomorrow: we should have colonies of Batch number four reporters on the plates we are transforming today.

• Glycerol Stock + Miniprep + Test Digest: reporters on Carb (tcpC(1),tcpC(2),tcpD(1),tcpD(2),tcpH(1),tcpH(2),toT(1),toxT(2),ctxA(1),ctxA(2),ctxA(3),tcpA(1),tcpA(2) + ctxB)
• Inoculate Batch nubmer three Reporters + Promoter (Chlora)
• Inoculate toxT gRNA.
• Ligate, Transform Batch number four Reporters + Promoter (Chlora)
• Anneal gRNA primers tcpF, acfA, acfB, Gel purify, Over-Night Ligation

Gel for annealed primers and gel purify later: tcpF, acfA, acfB.

Test digest. Doesn't look really good.