PCR questions...

Deanne Bell dbell at fresno.ars.usda.gov
Mon Apr 1 11:36:18 EST 2002

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".
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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