I am getting non specific PCR, I have tried with increase temperature and decreasing primer concentration but still non specificity is there? What parameters should I consider now?
Hi Ajeet,
I suggest before you try your next PCR enter your primer sequence into NCBI primer blast ( http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) and check in data bank for possible products correlating with your PCR product such as similar sequences, pseudo-genes etc.
After you rule this out all the above suggestion are worth while
Are you getting single nonspecific band or are you getting desired product as well?
Hello Drexler,
I have decreased amount of genomic DNA but there was no band in that condition. I have used 1.5ng/microlitre final conc of template DNA with Taq ploymerase, and it was bacterial genomic DNA.
Have you tried touchdown PCR? You can start with 72C or 68C (depending on which PCR enzyme you are using) for your annealing temperature and then decrease the annealing temperature by 0.5C per cycle and down to your target annealing temperature. Keep running PCR cycle with the target annealing temperature for another 20-25 cycles to get enough PCR product.
Or you can add different percentages of DMSO/PCR enhancer to your PCR reaction to reduce non-specific amplification. I usually try with touchdown PCR first and try with DMSO. If nothing works, you may need to redesign another pair of primer.
Hope this help.
@Thanks to all of you for guiding me....
I am gonna to include those ideas and let u know about my amplification.
Hi Ajeet, have a look here. Here are a lot of good solutions: http://www.bio-rad.com/de-de/applications-technologies/pcr-troubleshooting
Cheers, Nadine
Hi Ajeet,
I suggest before you try your next PCR enter your primer sequence into NCBI primer blast ( http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) and check in data bank for possible products correlating with your PCR product such as similar sequences, pseudo-genes etc.
After you rule this out all the above suggestion are worth while
Are you getting single nonspecific band or are you getting desired product as well?
The best thing is to design new primers. Seriously, some primers combined with unknown DNA/RNA and sometimes is necessary to do it or to rearrange old primers. Like Mr Ehud said blast in both, primer design and evaluation of created methodology is highly invaluable. Good luck! And remember PCR is "black magic" that we have to dill with :)
Hi Ajeet,
as Ehud alluded, are you sure that your primers are specific for your gene of interest. I would definitely blast them to make sure that they are specific for your gene of interest. It is quite possible that you will need to design new primers that are more specific and do not amplify other genes.
Try specifity amending chemicals (DMSO was already mentioned; I believe Betaine is also a good call).
Try different polymerases; in my experience, one sample can differ DRASTICALLY analyzed with several polymerases/kits, as the enzyme's specificity, productivity, processivity, and half-life differ.
Marcin - how true. E.g. Kapa Biosystem's HiFi DNA polymerase seems to be much more aggressive than many others out there. Also Invitrogen or Quanta kits perform much differently than the correlate AB kits. It seems one must eventually choose amongst the lesser of 50+ evils to eventually arrive at the system one wishes to perform through. And, once the system is identified, hang on, it could still be a bumpy ride.
You can try various methods.
Try to use a high fidelity enzyme. Phusion polymerase is the best from my experience.
And / Or
Do 1)an MgCl2 gradient
2) increase annealing temperature
3) decrease the amount of enzyme
4) decrease the number of cycles
5) if it a GC rich region , try a DMSO gradient 3-10%
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