Calvino Ka-wing Cheng
ckwcheng at acs4.acs.ucalgary.ca
Thu Jun 27 17:55:12 EST 1996
Hi again. Thanks for all your help the last time I posted a
primer question, it made life and sleeping a lot easier. We have
the primer figured out now, but we are wondering why we are
getting no PCR still.
We have tried various magnesium concentrations, to no
avail and we have tried doing PCR many times. Initially, we got
a VERY VERY VERY VERY VERY VERY dilute amount of amplified DNA
and now we are unable to reproduce that result.
1. We are wondering how our primers are. They were dissolved in
distilled water, making me wonder if they are still intact (due
to DNAse activity). How can we check? If they are not intact,
how do we tell?
2. Someone else defrosted the fridge and the PCR kit got up to
room temperature for I don't know how long. Which enzymes will
lose activity during this defrost cycle?
Taq? Pfu? Taq Extender (proofreading)? T4 Kinase?
3. Is there any way to tell that a primer has been made
incorrectly? (ie. is there spurious stuff inside it?)
Thanks. Your help is appreciated.
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