I am failing to obtain gel electrophoresis bands from PCR product of transgenic Wheat samples. There is always band present in positive control, but not in the wheat DNA samples.
I have tried increasing Mg concentration as well as DNA concentration but no results.
I have also tried a gradient PCR with no results except for in positive control.
Can anybody suggest how I should proceed further.
Is there a possibility for contamination in DNA samples inhibiting PCR amplification?
Can anybody suggest a good Wheat leaf DNA extraction protocol to try.
Dear Zain Tariq ,
Did you check for the DNA concentration and purity using Nanodrop or Qubit? I would suggest you look at your yield and purity of DNA before proceeding. If there appears to be impurities you could either try diluting the DNA 1:10, 1:20, to even 1:100 to eliminate the effects of PCR inhibitors. Alternatively you can try and purify the extracted DNA with a kit like the Monarch kits from New England Biolabs (they offer sample/tester packs) or the DNA Clean and Concentrator kits.
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Ironically, sometime adding LESS of the DNA will give you better results. If there are PCR-inhibiting molecules in the DNA, diluting your samples will give you better data.
Try a 1:10, 1:100, 1:1000 dilution of your wheat DNA & use a set of primers you know gives bright bands. Include a full set of controls.
Good luck!
Your positive control is giving the results which means all the reagent and parameters are working well. I would suggest , check the purity of your template DNA. Try running your DNA on 0.8% Agarose gel to see if DNA is present or not( if not using nanodrop) . Sometimes , If the DNA is too concentrated it might not give you the result. I would recommend make the dilutions of template DNA 1:10 , 1:50 or 1:100 work well.
On the otherhand, very less DNA quantity can also give you negative results.if you are using a manual porotocol try using qiagen DNA extraction kit or anyother kit at your hand it might resolve issue in your DNA yeild.
Also check the primers you are using are well optimized or not.
Hope this helps
Good luck
I would suggest trying to design new primers, or performing nested PCR, the conditions of your PCR reaction may be not working for the primers you have.
I would suggest you ethanol precipitate your samples or clean them up using a commercial option because it sounds like a contaminant got in and is inhibiting your polymerase. Alternatively, and more expensively, Platinum II from Invitrogen is my professional opinion cannot be stopped. I don't know what they did to make that polymerase but I have never had a PCR fail when I use Platinum II in my +10 years of research.
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