I'm using a two markers RG140/PvUII and s10019/BstUI to detect fertility restorer genes in Oryza sativa. I optimized the protocol, now there is a problem with amplification that the fragment stay inside the well. The gel is depicted here; note I also ran it on agarose low concentration gel, also used the new primer from stock primer.
I have extracted DNA from various templates, from medicinal plants mostly, which all have secondary compounds that interfere with DNA extractions and consequently with PCR amplifications. My guess is that your template has compounds in it that prevent a PCR reaction, or you are using too much template DNA. Have you run your genomic DNA on a gel to check quality (distinct band on top of the gel)? How much do you use for a PCR reaction? I found that 10-20ng is sufficient for a 25uL reaction.
I too suggest to always include a positive control (primers that are known to bind to the template DNA, ITS region or so) and a negative control (mastermix without DNA, to see if supplies are without a contamination).
Check your loading buffer by mixing it with DNA that you know the size (plasmid or genomic) and run it on a gel to see if stays in the well, then you know your loading buffer is bad.
Interesting. How can you say the amplification results are positive? Have you a positive control? If not, in the next amplification, use a sample with ubiquitine primers as positive control.
In second place, the ladder is very pale. Are you sure your electrophoresis buffer is not contaminated? Try to change it and re-run the gel, maybe adding a dna quantity of which you are sure, as second positive control.
I have extracted DNA from various templates, from medicinal plants mostly, which all have secondary compounds that interfere with DNA extractions and consequently with PCR amplifications. My guess is that your template has compounds in it that prevent a PCR reaction, or you are using too much template DNA. Have you run your genomic DNA on a gel to check quality (distinct band on top of the gel)? How much do you use for a PCR reaction? I found that 10-20ng is sufficient for a 25uL reaction.
I too suggest to always include a positive control (primers that are known to bind to the template DNA, ITS region or so) and a negative control (mastermix without DNA, to see if supplies are without a contamination).
Check your loading buffer by mixing it with DNA that you know the size (plasmid or genomic) and run it on a gel to see if stays in the well, then you know your loading buffer is bad.
Hi!
Few months back Jh Fan asked the same question thinking the junk in the wells as PCR product. Of course compare to his gel image your gel picture is looking clean and appearing the lower line of the well as PCR product.
Refer the following link you get useful answers.
https://www.researchgate.net/post/PCR_products_stick_in_gel_wells_PCR_problem
By watching your gel image I say your PCR did not work. Marker also looking feeble.
Refer literature and repeat PCR carefully, hope you are using correct Ta and all your PCR ingredients are intact including template DNA. Certainly it should work. Safe side put +ve control your doubt will be crystal clear is it PCR problem or DNA problem?
Good Luck!!!!
The result of Primer RG140 is a monomeric fragment of 1500 bp (the restriction enzyme (PvuII) will show polymorphism) for all sample and I get it in previous PCR showed polymorphism . Now the problem arise although i repeated it several time and i get the same results with template dilution.
Thanks to all for suggestion, i will try and follow the ideas.
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