Cloning PCR fragents into vector

Mark Routbort 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.
Phenol/CHCl3/EtOH.
   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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