(2pts) Basic steps to clone a gene into a vector for expression: (1)PCRamplificationofthe gene, (2) digest (cut) both the double-stranded PCR product (insert) and plasmid vector with the same restriction enzyme(s) to create complementary "sticky ends", (3) allow the digested insert and vector to anneal, and (4) ligate the annealed fragments together with DNA Ligase.
A. What additional step is required to generate a DNA template for PCR from a eukaryotic organism rather than a prokaryotic organism, and why?
B. Why is it necessary to re-circularize (ligate) a plasmid prior to transformation into cells?
A) Splicing and... View the full answer
- b) Reasons - 1) Only circular DNA molecules are able to replicate, may confer antibiotic resistance to the bacteria whereas Linear DNA will not replicate (and will not survive exonuclease activities) inside the bacterial cell 2) Transformation efficiency is lower for linear DNA fragments compared to circular DNA 3) Linear DNA are subjected to degradation by exonucleases inside the cell
- May 12, 2018 at 5:02am