So, here is what I am dealing with:
The Tm of the primers is 55 for the forward one and 56 for the reverse one. In -silico PCR produces 750 BP band, but in reality I get 3 bands. The third one looks like a primer-dimer.
Clearly the primers could be decreased and I used primers diluted to 10 uM each and I 1:3 dilution from it, which didn't give me any product. I tried 1:2 dilution today: this decreased band intensity and didn't get rid of the extra/lower band.
The question is where do I go from here?
-Gradually raise the annealing temperature?
-Will DMSO help?
-Increasing DNA concentration, while decreasing primer concentration?
Does Tm really matter? Should I just try different annealing temperatures?
Thanks in advance !
Hi, there are some possibilities in getting non-specific PCR bands:
1) Primer problems, like high -ve energy for primer dimer, self-dimer etc.
2) Annealing temperature problem. Maybe you can run your PCR at different annealing temperature first, for example 50, 52, 54, 56, 58 and 60 degree celsius and try to see at which temperature would give you better band intensity and less problems, eg less smearing and absent of non-specific band
3) Contamination issue, eg your extracted DNA contains another DNA etc.
Try to think back about the whole process from DNA extraction, primer design, how you run the PCR and gel electrophoresis and hopefully can troubleshoot your problem.
Good luck.
Regards,
Chong
Hi Leonid,
It sounds as if you are having issues with specificity and/or possible contamination.
From a specificity perspective the above steps you mentioned with regards to gradually raising the annealing temperature and decreasing the primer concentration are good first steps.
I would also consider using a hot-start DNA polymerase for increased specificity if you are not already using one. (I'd be happy to recommend if you require more information).
If contamination is a potential concern, I would suggest following some of the steps in the link below, I have found this to be very useful in the past:
https://bitesizebio.com/20773/clean-up-your-act-how-to-clean-up-pcr-contamination/
I am pretty sure I don't have a contamination, but I can double-check. This primer is from a publication and they did use hot start polymerase, but I don't have it right now. Interestingly, the conditions they used did not give any results.
It is not that important right now that I have extra bands, but I'll keep working at it.
Thanks for the input.
if you want to do a hot start pcr but do not have the enzyme then do it the old fashioned way. Mix all reaagents except the enzyme. Hold the mix at 80c in the cycler. Manually add the enzyme 1n 1ul of 1x buffer and immediately start the cycling. The temperature will never drop below the primer annealing temperature so primer dimer is minimised. Works fine for small numbers of samples.Use new tip for each addition to stop contamination
Thanks, but not possible-I am using a ready mastermix :) If I get hold of polymerase separately, I'll surely use your suggestion!
it is still possible, withholding Mg if it is added separately works just as well or possibly even adding one primer to the hot mix may work but the Mg is second best after the enzyme
Unfortunately, it also contains Mg 2+. I have asked another lab to lend me some polymerase and will do PCR tonight. Thank you !
Hi Leonid,
I think you must check your primer. your primer may detect other unwanted target.
check ur primer at primer-blast
https://www.ncbi.nlm.nih.gov/tools/primer-blast/
So, you can extract your expected band from gel by gel extraction kit and then send it for sequencing.
It is for determination.
I suggest maybe you should increase your annealing temperature, or run pcr with various Ta. But I ever heard, to get specific band, you can vary your denaturation and annealing temperature. If there's not problem with your primer or your pcr and you want your specific target, you can purified your pcr, using column or gel purification
you could consider nested internal primers and a re amplification of your dirty product which often cleans up an amplimer.
If the molecular size of third band is relatively low (around 100 bp) there is a big chance its a primer dimer! You may try to use negative control, then all bands will be not amplified from your DNA sample!
Best luck
OK, so I wanted to give you an update on this: I assembled a PCR reaction using all ingredients separately, as opposed to using the mastermix I had. As Paul Rutland suggested, I added polymerase into the reaction last, during the 10 min incubation at 95C , after which the program switched immediately to cycling. This did considerably help. The primer-dimer is gone and there is only a faint extra band, but I'll figure out how to get rid of that one.
Extra band? Do you mean multiple bands? If that so, please optimise your denaturation and annealing temperature. If you want to sequencing one of specific band, you can purified your pcr product.
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