BIO370Y5 Lecture Notes - Lecture 1: Sticky And Blunt Ends, Dna Supercoil, Ethidium Bromide
Document Summary
The goal of labs 1-4 is to reconstruct a recombinant plasmid with ampicillin (from pamp) and kanamycin (from pkan) In part a, a restriction digest of plasmids pamp and pkan occur: they are digested with hindiii and bamhi (then incubated in 37 c, digested plasmids are placed in 0. 8% agarose gel to check to see if it worked. Lab 1: the buffer is used to maintain the ph of the bacteria so they do not die. The 2x is just the different ionic strength needed, and is used to keep the restriction enzymes stable. In terms of the amount of buffer you add, just make sure it does not go over the top cause then the dna will run through the buffer and not through the gel. We only use a small amount of digested pamp and pkan to check if it actually did get cut. Lambda dna is digested with bsteii and you get your standard curve from it.
