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Trouble with PCR on genomic tomato DNA (I've tried many fixes)


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#1 Tomato

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Posted 23 July 2009 - 11:52 AM

I am working on a tomato project that requires DNA to be isolated from tomato plants and then analyzed using primers that target the SW-5 gene. The primers come from previously published work, and are known to generally work well. I have made sure that the DNA quality and quantity are both good using a nano-drop instrument. I am expecting a product that is ~750bp. The PCR product will most frequently show short products, most of the time they appear as a smeared band from ~50bp-400bp, rather than a discrete band at a specific length. Do primer dimers usually occure as smears?

I have tried adding DMSO, Betaine, and BSA, with no improvement to the product. I have also tried using different reagents, I have had new primers made, I have tried hot start reactions. I have tried adjusting the ammount of MgCl2, KCl, Taq, and dNTPs. I have used a gene clean turbo kit to attempt to remove any polyphenols etc that could be inhibiting PCR.

I keep getting these short smears when I run gels. I have used control DNA and control primers to ensure that the thermocycler works propperly, once yielding exactly the product it was supposed to and more recently giving me the expected product in addition to smears that are the same as I am getting when I try to use the tomato DNA and tomato primers.

This is work that has been published in many papers with no problems. I am following their protocols exactly. Any suggestions to what might be going on with my attempt to amplify my gene of interest? Thanks

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#2 leelee

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Posted 23 July 2009 - 07:54 PM

are you using the same enzyme as the published groups too??

#3 phage434

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Posted 24 July 2009 - 07:53 PM

I'm guessing that you are using too much genomic dna template, which can (because of inhibitors) actually be less effective than lower amounts. Try 10x and 100x dilutions of your genomic DNA.

#4 Tomato

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Posted 29 July 2009 - 11:43 AM

I tried both 10x and 100x dilutions with absolutely no improvement to the PCR. I'm currently trying different extraction techniques. I have noticed that the Genomic DNA appears to have much more short fragments than long fragments when I extract it and observed in a gel. Maybe this could be the reason? But even still there is some very long DNA, I would think that this would give me at least some of my expected product?

#5 phage434

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Posted 29 July 2009 - 06:33 PM

How are you isolating the gDNA? Plants often have phenols present that co-precipitate. You might need to try a CTAB isolation procedure to eliminate these contaminants.

#6 Tomato

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Posted 29 July 2009 - 06:54 PM

Yes, I have tried CTAB, I more frequently use a miniprep protocol that has seemed to give better isolation. More recently I have been trying samples that various companies have sent me that are specific for isolation of Plant DNA with high levels of the potential PCR inhibitors that you are talking about. I tried a new extraction method today, a plant DNA isolation method from epibio. that does not require any grinding or physical lysing of the leaf sample. I will try PCR with the new extract in a day or two.




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