environmental DNA gels

David Johnston dmj7 at bellsouth.net
Sun Feb 6 17:31:43 EST 2000


On 2 Feb 2000 14:32:46 -0000, dharvey at ocean.ocean.fsu.edu (Danielle
Harvey) wrote:

>Hi all,

Hi Danielle!!

>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

A picture of the gel would help... but off the bat, I would suggest
that it's your PCR reaction amplification conditions, not your gel
conditions, that are causing your smearing.  A260/280 ratios are not
really relevant for PCR on genomic DNA these days:  it tells you about
contaminating proteins and RNA that really do not adversely affect
amplification, but does *not* tell you about the presence of PCR
inhibitory substances.   Therefore, you may want to examine your DNA
extraction procedure, and maybe as a control use a Qiagen DNA column
and see what that does.  

Having said that, I'm almost positive it's your reaction conditions--
either the primer sequences, annealing temp/time, and extension time.
For amplitaq gold, allow about 30 sec./kb of amplicon.  If you're
getting smearing, suggesting some mispriming, try raising your
annealing temp in 2-3 degree increments.  Also, with degenerate
primers, it's smart to have a slightly longer annealing time-- but
most people grossly overdo annealing times anyway.  

I'd be more than happy to take a look at your protocol and give
suggestions if you email me Monday.  Our facility develops PCR assays
and performs over 1 million rxns a year.

Good luck!

Dr. David M. Johnston
johnstd at labcorp.com







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