Cloning PCR fragents into vector
rout at quads.uchicago.edu
Wed Sep 12 09:39:37 EST 1990
I'm looking for someone with experience cloning medium sized (500-1000 bp)
PCR fragments into a vector for screening. I'm having troubles getting
transformants to screen.
I discovered through a control experiment that Bluescript vector that I
had cut, blunt-ended with T4 polymerase, phosphatased with CI alkaline
phosphatase, and then kinased with T4 polynucletide kinase would NOT yield
Amp resistant colonies. Unfortunately I don't have the cut, blunted,
unCIAPed vector to determine if the problem lies with blunting or with
kinasing (although either is a problem, since I have to both blunt and kinase
the PCR fragements).
The sequence of steps on the above control was:
1) Cut CsCl-pure DNA (of a clone that has a 3 Kb insertion into SmaI site of
Bluescript) with Asp718I, XbaI, and StuI (which cuts not in vector but in
insert so I could distinguish vector fragment from insert). phenol/CHCl3/EtOH.
2) T4 polymerase to hopefully blunt. phenol/CHCl3/EtOH.
3) Calf intestinal alkaline phosphatase. Heat inactivate in presence of
EDTA (15' at 65 deg C). Prep gel. Cut out and elute 3 Kb Bluescript fragment.
4) Rephosphorylate with T4 polynuc. kinase.
5) Use 100 ng of DNA in kinase buffer to ligate with 10 units T4 ligase
overnight at 12 deg C.
6) Transform into XL-1 competent cells. No colonies next day.
I've never had problems ligating or transforming before using cohesive or blunt
ends (directly from enzymes), so I think the problem lies either with the
kinase or the polymerase (both brand new). Has anyone had similar problems and
resolved them? Unfortunately I _can't_ cut the PCR fragment and ligated with
cohesive ends on both sides, although I can do so on _one_ side.
Thanks for any tips, insights, and sympathetic commiserations.
Mark Routbort Due to circumstances beyond my control,
rout at midway.uchicago.edu There will be no big parade this Sunday.
-- Colonel Scheisskopf in Catch-22
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