Purifying clean plasmid vectors for ligation

Harry J. Witchel Harry.Witchel at bristol.ac.uk
Wed Mar 13 08:18:20 EST 1996


>>  ...
>>         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).


>Is it possible that enzymes/buffers for PstI and XhoI have some
>contaminant that is active against single-stranded overhangs?
>
I suspect that the problem is with the gel purification and not with my 
digestions for the following reason:

If I just do the digestion followed by the phosphatase (but without any gel 
purification), the kinase control works perfectly (the plasmid can 
recircularize), but unfortunately the negative control (ligation without 
kinase) produces approx. 1:20 transformants as the kinase control, so with 25 
ng of vector, I get 40 or so colonies .  As I am looking for 400 bp inserts 
which are often blue colonies anyway, the background is very high for 
screening.  It is for this reason that I tried to gel purify it.

	Nevertheless, I will look into problems with my enzymes and buffers.
Thanks, 
	Harry




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