i have ligated 40 kb fragment in PCCFOSII vector. now i want to confirm ligation without wasting packaging phage particles. can i transform my ligated product in DH5alpha. Kindly suggest me, if any of you have done the same experiment.
Unfortunately, transforming in DH5alpha will not work owing to its size. We have used epicentre Phage kits for our mycobacteria work where we have ligated approx 55-58 kb fragments. So the way we do our expt is to go for a 16C ligation for 16 hrs. We use twice the amount of Ligase (Neb) or Conc Ligase. We use half the packaging extract per reaction with half the amount of ligated DNA. So Checking alternatively is not an option to try. I am unaware if someone tried electroporation (Since size does not matter here). Atleast I have not tried. BUt Chemical transformation will not work.
Hope it helps
Dear Sathya narayan,
Thanks for your reply. using half of the packaging extract is good idea.
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