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-   -   genomic DNA PCR: Troubleshooting/General Questions (http://www.molecularstation.com/forum/pcr-polymerase-chain-reaction-forum/47622-genomic-dna-pcr-troubleshooting-general-questions.html)

ChameleonAmy 12-07-2008 06:54 PM

genomic DNA PCR: Troubleshooting/General Questions
 
Hi there, I'm a newbie at molecular biology -- so my apologies for any completely naive questions that follow!

I isolated genomic DNA from a ~5Mbp prokaryote. I am interested in amplifying ~1.5kbp sequences from this genomic DNA. Unfortunately, I don't know the exact sequence of primers to use for this amplification since there's no sequence data available for this particular microbe. -- However, there is a strong literature precedent for success using specific degenerate primers, so I have been trying to use those. I am also using the PCR conditions previously reported in the literature (Taq polymerase, with recommended concentrations of MgCl2, DMSO, etc.). Unfortunately, I am not seeing any amplification in my PCRs with this genomic DNA.

In my efforts to figure out what's going wrong here, I am wondering about the following:
-Was the amount of genomic DNA used in my PCRs sufficient for amplification? I estimate I used ~60ng of this ~5 Mbp gDNA in each 50uL PCR. Might it be useful to increase the amount of gDNA in each reaction? But, this would require increasing the amount of gDNA mixture added to the reactions, and this begs the question:
-At what concentration does the EDTA in TE become 'toxic' to Taq? My gDNA is currently dissolved in TE, and I have been using 1uL of this mixture in my 50uL volume PCRs (My "stock" conc. of gDNA in TE is ~60ng/uL). Could this amount of TE be inhibiting Taq? And, how much TE could I reasonably add to the PCR?
-How specific are Taq-catalyzed PCRs? -- In other words, I am wondering: if my [degenerate] 16-20bp primers are mismatched by 2-3bp from the template genomic DNA, should PCR still work or not?

Thanks for any and all advice. As a chemist dabbling in mo bio, I very much appreciate any solutions to my frustrations! :)

butters 12-12-2008 06:38 PM

Re: genomic DNA PCR: Troubleshooting/General Questions
 
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question1
- amount to load 60ng seem to be ok
question2
-check the above link; by the way what is concentration TE are u using?
question3
-it is degenerative primer, sure it work. (if presence of mismatch is not at the end of 3' end.)

if you can provide info below we could help up more on what is the pcr condition used?
-cycle number
-Tm
-etc

Advice;
Well we will try to help out but sometimes it is best to contact the corresponding author for detail especially if you have tried all the parameter mentioned in the paper. if there is not response from the author could u give us the paper and we have a look. and try to guide you the best we could

:D cheers

chibneelam 01-08-2010 11:21 AM

Re: genomic DNA PCR: Troubleshooting/General Questions
 
i want to know what actually degerative primers are?

butters 01-12-2010 03:55 PM

Re: genomic DNA PCR: Troubleshooting/General Questions
 
@chibneelam, its degenerative primer. As far as I can simplify it with my shallow knowledge. Degenerative primer is mixture of primers that contain slight variation to amplify a group of target gene regardless. It is somewhat a universal primer.

I would suggest you google it and read up :)


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