On Thu, 3 Oct 1996, Duncan Greig wrote:
> I am trying to PCR up a 2kb fragment, and am having difficulty getting it
> to appear using colony PCR I have had good results with the primers after
> making DNA, but it would be convinient to be able to use colonies.
>> Does anyone have a protocol that has produced such large fragments reliably?
>> thanks.
>> Aidan Weatherill.
>
Dear Greig:
There are two ways: 1) boil colony which should be picked with pipet tips
in 1XPCR buffers for 5min; 2)incubate colonies in small amount of
zymolyase solution for 5min. After the either way of breaking yeast
cells, you could do PCR as usual. It works fine. By the way, I have
used Pfu polymerase and its buffer (no affiliation to Stratagene), and I
just haven't tested with other polymerases. Theoretically there should
be no problem with others. Cheers!
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