environmental DNA gels

Danielle Harvey dharvey at ocean.ocean.fsu.edu
Wed Feb 2 09:32:46 EST 2000


Hi all,

I am getting a lot of primer dimer, mispriming (unexpected fragment
lengths) and smearing in my PCR rxn and agarose gels using degenerate
primers for nitrogenase (nifH) and nitrate reductase (nirS, nirK) and
Taq Gold polymerase.  I am working with extracted DNA from marine
sediments that is pretty pure, based on and A260/A280 ratio of ~ 1.8.  I
have just switched from ethidium bromide staining to SYBR Gold and still
see a lot of smearing.  I was wondering if anyone had any suggestions as
how to acheive clean-looking gels.  My gel protocol is 84 volts for ~
1.5 hrs with 1% TBE agarose and a 2X TBE running buffer.  Any
suggestions would be helpful.
Thanks,
Danielle
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