Basic Cloning
PCR ins-F ins-R insulin_cDNA pcr_ins
Digest pcr_ins NcoI,XhoI 1 pcr_dig
Digest pET28a NcoI,XhoI 1 vec_dig
Ligate pcr_dig vec_dig lig
Transform lig Mach1 Kan 37 pET-INS
Five lines, where Golden Gate will take one. PCR the insert with primers that carry the two sites, cut it and cut the vector with the same pair, ligate, transform. This is the construction file from the basic cloning tutorial, the same one you will run, so read it there as well. Notice the number on each Digest line: cutting with two enzymes leaves three pieces, and you have to say which one you want. That bookkeeping is the thing the standardized methods later in this lecture are trying to take off your hands.
Section divider: Basic Cloning. Beneath it, the Construction File for the experiment, in monospace, five lines: PCR the insulin cDNA with primers ins-F and ins-R to give pcr_ins; Digest pcr_ins with NcoI and XhoI, keeping fragment 1, to give pcr_dig; Digest pET28a the same way to give vec_dig; Ligate the two digests to give lig; Transform lig into Mach1 on kanamycin at 37 degrees to give pET-INS.