Thursday, June 08, 2006

Ligation, Transformation, Update

PCR with 35 cycles was perfect. Details of run are in previous post.

I liagted the fragment yesterday (Wednesday) into the Novagen pETBlue-2 vector. I deactivated the Taq with chloroform and used 2 ul of the aqueous phase in the ligation. The fragment was blunted then ligated with T4 ligase. I transformed NovaBlue Singles (a K-12 derivative) and plated on LB with carbenicillin, tetracycline, X-gal and IPTG.

Today (Thursday) all the test insert cells were white and all test plasmid colonies were blue, as hoped for. I didn't have many colonies on the plate with the IDP2 plasmid. Nonetheless, the few I had were white. (I have more of the cells unplated [still in the tube that I transformed the cells in] stored at -86 C, in case I need to try replating the transformants. Each tube has 250 ul of SOC.) I picked six of the colonies and innoculated 3 ml TB cultures. The cultures are now in the incubator at 37 C with shaking at about 220 rpms.

Tomorrow, as long as the cultures are saturated, I will do minipreps on the six cultures, then do restriction digestions, then run on a gel.

Fingers crossed!


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