PCR questions...

[Deanne Bell]

Dear NM
:
:1) Will MgCl 'go off' after repeateded freeze/thaws??
Are you talking about PCR buffer purchased with the MgCl already in it?
I am compulsive about purchasing MgCl separate from my 10X buffer.  Both items are shipped frozen - I heat the MgCl at 65*C for an hour or so to make sure there are no crystals and then store it at 4*C.  I keep the buffer at -20*C until just before each use.

:2) Is it common to do a PCR without doing a hot start and see nothing,
:but then get a great product using a hot start??? It seems unlikely
:that it would go from nothing to something under these conditions...
:by the way, the product size is 1100bp...
:

Are you using the same polymerase for the hot vs. "not-hot" start?
How are you doing the hot-start?
If you use different poymerases, you can "go from nothing to something" due to MgCl interactions or DNA "preferences".
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Deanne Bell
USDA Agricultural Research Service
9611 South Riverbend Avenue
Parlier, CA 93648
(559) 596-2806  voice
(559) 596-2791  fax
dbell at fresno.ars.usda.gov
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