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 
confusing.

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 
agarose).
	Thanks one and all,
	Harry




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