Lab module 2 - conjugation

Question 1

plasmid mini-prep

Answer 1

inoculate LB with E. coli containing the vectors.
use plasmid mini-prep kit (alkaline-lysis method) to isolate highly purified plasmid DNA. the method involves breaking open the bacterial cells using a combination of lysozyme, which breaks down the cell walls, and SDS, which complexes with the proteins. Sodium hydroxide is included in the lysis buffer and the resulted high pH causes DNA to become single-stranded. The mixture is then brought back to a neutral pH by adding the neutralisation buffer, and this causes precipitation of chromosomal DNA, proteins, and other cellular components, but leaves the plasmid DNA in solution. The precipitate is then removed from the mixture by centrifuging. The plasmid is then recovered by centrifuging the supernatent through a column. The column filter contains a silica surface. The plasmid DNA absorbs to the silica surface while contaminants are passed through without absorption. The plasmid DNA is then washed in desalting buffers and sterile water.

Question 2

restriction of plasmid DNA

Answer 2

dna treated with restriction enzyme BamHI and then visualised using agarose gel. This will hopefully show that an extra fragment of DNA is present in the clone