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
>> 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
>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.
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