Image: first lane is 100bp marker, followed by different DNA samples apmlified with same primer pair (conventional method used for dna extration). last lane DNA amplified with same primer pair and product size is as expected (kit was used for extraction - last lane)
PCR condition is already optimized as i am getting product for genomic DNA extracted using kit but not for genomic DNA extracted using conventional method.
why am i getting smear?
Hello Yash,
It seems that there is no problem in PCR conditions as you got band from the DNA isolated by kit. Check the quality and quantity of the DNA isolated by conventional method. There may be some impurities in DNA which hinder the PCR amplification. Give treatment of RNAse and Proteinase K to the isolated DNA and try again to amplify.
Hi Ravinder,
During DNA extraction, RNAse and Proteinase K treatment is already given to the DNA. As you said, I am also assuming that, the DNA must be having PCR inhibitors. I have tried PCR after diluting DNA, but result is same. I will try PCR with different DNA dilution.
Thanks
Yash
I would NOT say that your PCR is "optimized". Your positive control band is rather faint. Have you used gradient PCR to find the optimum annealing temp? That's usually the limiting factor in PCR.
Did you verify that your DNA extractions worked using some sort of robust primer pair (actin or similar)? Or test the concentration with a nanadrop or Qbit? If you have inhibitors, less DNA usually works better than more DNA.
Good luck!
hello Katie,
I have used gradient PCR to find optimum annealing temp as same primer is giving bands with DNA extracted using kit from different individuals.
I have checked DNA concentration using nanodrop, and quantity and quality looks good.
I would consider to use robust primer before using newly developed primer.
Thank you.
Hello Yash,
Apparently the problem is with the conventional extraction method you have used. How was the 260/230 ratio?. Your DNA may contain PCR inhibitors that could possibly affect the successful amplification.
Did you check your DNA on a gel for the integrity before PCR? Fragmented DNA or contamination can give this kind of smears.
Thank you everyone for suggestions.
I came to conclusion after checking all parameters, The some sample has less DNA concentration (but good quality -- less degradation )and must be having high PCR inhibitors.
Is there any protocol to amplify DNA having high inhibitors ?
Reasons can be:
1. bad DNA quality
Check the quality of DNA with "universal" primers ( the primers that can anneal with DNA of almost every species) or with primers that you know they must amplify the product on your DNA
2. you did not write anything about the samples. What makes the last sample different from others, except DNA isolation? Maybe the selected SSR primers can not link to your DNA so you need to select new one
Many thanks everyone for suggestion,
DNA samples are from different individuals of house cricket insect. last Sample DNA isolated using kit from different individual.
After troubleshoot
1. DNA quality is good enough for PCR (Checked with nanodrop)
2. DNA samples amplified with universal primer and i am getting PCR product.
3. The ABS is used to reduce effect of PCR inhibitors effect of amplififcation.
4. I have checked many SSR primers (screening is in progress) and some of them are not amplifying the DNA at all from different individual.
Regards and many thanks.
Yash
4. I have checked many SSR primers (screening is in progress) and some of them are not amplifying the DNA at all from different individual.
but some of the primers gave the PCR products. It confirms my second doubt: selected SSR primers can not link to your DNA (despite other researchers get PCR products, even on samples of same species). So you need to select new
Dear Ksenija Taski-Ajdukovic,
Yes, you are right about it, The newly developed primer pairs screening in progress for amplification. I have developed SSR markers using newly sequenced genome. I have made primer library and using it for screening.
Thank you very much for prompt responses.
Regards,
Yash
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