Purifying clean plasmid vectors for ligation
Harry J. Witchel
Harry.Witchel at bristol.ac.uk
Mon Mar 11 06:22:33 EST 1996
Salutations Molecular Soul-brothers and soul-sisters --
I have had mixed success purifying plasmid vectors which have been
linearized, treated with phosphatase, and gel purified. I have run the
purifications side by side, so the fact that the results are mixed is very
THE PROTOCOL: Qiagen maxi-prepped plasmids were linearized using overnight
digestion in NEB enzymes with appropriate buffer. The reaction volume was
doubled, some phosphatase buffer was added, and a calf intestinal alkaline
phosphatase reaction was run for an hour. Phenol chloroform extract, then
chloroform extract. Ethanol precipitate. Run on ordinary agarose gel. Cut
out bands, electro-elute bands into a dialysis bag. Harvest buffer in
dialysis bag. Refrigerate, and spin out any agarose remaining in fluid.
Ethanol precipitate. Resuspend in TE, spin out insolubles, ethanol
precipitate, resuspend, spinout insolubles, ethanol precipitate, ethanol wash.
At this point the vectors are done.
TESTING THE VECTORS: After everything was finished, the vectors were run on
an agarose gel to check for purity and concentration. All three vectors were
100% pure, linearized, and they were concentrated at 30 ng/ul. The vectors
were tested functionally by autoligating them, either with or without kinase
pre-treatment. It was T4 PNK for one hour, phenol chloroform extract,
chloroform extract, then ethanol precipitate. Finally autoligations were
performed using 30 ng (1 ul) of each vector, using T4 ligase and BRL buffer,
which includes PEG.
REACTIONS WERE RUN SIDE BY SIDE: Three vectors were tested: pGEM 3zf- in PstI
and CIP, pGEM 3zf- in SacI and CIP, and pBluescript II sk+ in XhoI and CIP.
All three vectors were present at the end of the purification (as shown by the
gel). The alkaline phosphatase reactions were prepared in one master mix tube
and divided out onto the digested vectors. The kinase reactions were prepared
the same way. The ligase reactions were also made using master mixes.
RESULTS: When autoligated (recircularized) without any pre-treatment, all
three vectors made 0 colonies on AXI plates. When autoligated with kinase
pre-treatment, only pGEM SacI made any colonies (many colonies -- 40 in a
small percent of the total transformation); the other two vectors made no
colonies. Furthermore, when the pGEM pstI vector was ligated to a gel
purified (but untested) PstI 200 bp fragment, there were still no colonies.
Any ideas as to how I could have run three simultaneous reactions and
ended up with only one working? Any ideas as to how to get rid of agarose
contaminants from high melting point agarose (beside switching to LMP
Thanks one and all,
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