We tried the pea primers, but didn't get any result on our gel, so decided to run genomic DNA to test extraction.
Our write up is here. http://wiki.london.hackspace.org.uk/view/Plant_species_testing
We've done this a few times with different methods, and each time we get a band or smear always running ahead of the 2000 - 100bp ladder, whereas we would expect genomic DNA to run much slower. Methods we've tried are chelex extraction, isopropanol / ethanol extraction, and simple 'homogenisation' (crushing and mixing). Would be great to get some input of possible causes, things we've thought of are:
- DNAses in our reagents
- The fast bands are RNA
On Wednesday, 11 July 2012 02:01:16 UTC+1, Nathan McCorkle wrote:
On Mon, Jul 9, 2012 at 1:36 PM, Nathan McCorkle <nmz787@gmail.com> wrote:--
> I would start by vortexing a few times rather than the once at the
> end. You could also add freeze-thaw cycle(s) if you think lysis is the
> problem.
>
> Next I would do an isopropanol (70% v/v) extraction followed by an
> ethanol extraction (or 90% isopropanol if you can't get 90%+
> ethanol)... then let the tubes air dry for a while until the alcohol
> is gone... some people say its hard to redissolve DNA if it dries out
> all the way, some say they have no problem... but just dry them long
> enough that you don't smell alcohol.
>
Sorry, to be clear, I meant to do the alcohol extractions after the
chelex steps... since chelex is a chelator, if there are trace amounts
it could be soaking up the Mg2+ in the PCR mix and messing things up
there.
> Post-stain gels instead of running them with ethidium bromide, as it
> runs opposite DNA so it actually slows down your gel and can decrease
> resolution.
>
> Keep us updated!
>
> On Wed, Jun 27, 2012 at 7:12 PM, TRolandB <williambeaufoy@gmail.com> wrote:
>> Hi everyone, our group (London hackspace) have been doing some genetic tests
>> (sex typing) recently, with PCR and electrophoresis. We're getting very
>> inconsistent results, and think our extraction process is at fault, so we
>> want to try some similar techniques but with DNA from foodstuffs, so we can
>> be sure of getting the extraction right.
>>
>> Does anyone have any suggestions of tests you can do that involve PCR and
>> electrophoresis of this type?
>>
>> Will
>>
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>
>
> --
> Nathan McCorkle
> Rochester Institute of Technology
> College of Science, Biotechnology/Bioinformatics
--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
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