After doing a PCR with my specific primer I got multiple bands instead of getting a unique band. I am totally astonished after getting this. Can anyone guessed what happened why I get this band after PCR?
Seems that you are getting non specific amplicons. Resons could be :
1. the primer isnt uniquely specific and is partially binding to multiple sites ( Increasing the primer length may help increase specificity or do a gradient PCR to see if different annealing temperatures show a better profile or better yet, go for touch down PCR )
2. your primer is forming secondary structures (use 5% DMSO in your reaction)
3. there is foreign DNA contamination (use a no template negative control)
4. too much primer in the reaction. ( 0.1 to 0.2 µM should be more than enough)
5. the smears could mean that the agarose gel isnt fine or that the annealing time and temperature was high or elongation time was not enough (reduce 5 seconds of your current annealing time and try not to increase the temperature beyond what you have tried already and increase elongation time according to your polymerase's datasheet)
And try to reduce the DNA template. (1 to 10 ng for Genomic DNA and 1 to 10 pg for plasmid DNA per 50 ul reaction should be fine).
As you can see the possiblities are many. You can systematically try to troubleshoot one issue at a time till you can find the problem. All the best.
Seems that you are getting non specific amplicons. Resons could be :
1. the primer isnt uniquely specific and is partially binding to multiple sites ( Increasing the primer length may help increase specificity or do a gradient PCR to see if different annealing temperatures show a better profile or better yet, go for touch down PCR )
2. your primer is forming secondary structures (use 5% DMSO in your reaction)
3. there is foreign DNA contamination (use a no template negative control)
4. too much primer in the reaction. ( 0.1 to 0.2 µM should be more than enough)
5. the smears could mean that the agarose gel isnt fine or that the annealing time and temperature was high or elongation time was not enough (reduce 5 seconds of your current annealing time and try not to increase the temperature beyond what you have tried already and increase elongation time according to your polymerase's datasheet)
And try to reduce the DNA template. (1 to 10 ng for Genomic DNA and 1 to 10 pg for plasmid DNA per 50 ul reaction should be fine).
As you can see the possiblities are many. You can systematically try to troubleshoot one issue at a time till you can find the problem. All the best.
Hi,
I agree with dr. Praveesh Valissery and dr. Ruslan Kalendar.
I would recommend you to run always with the marker (GelRuler) first of all because I do not see a reference in here. The multiple bands might be due to the non-optimal PCR conditions so you should adjust your annealing temperature (try with a gradient at first), optimize the DNA extraction (might have some contamination, you can easily check with an agarose gel run) and then I would suggest you to dilute your DNA and primers (1/10 dilution should be ideal).
If the primers might show still problems, you shall as well try with a Hot-Start.
Best regards
First solution - if possible go for nested primer. It would work like magic. If not possible try touchdown pcr. It works for some people. But i would advise you to change primer, mastermix (change to hotstart from promega. Its the best)
There is the possibility that the annealing temperature is too low. You can increase it and even go for a temperature gradient.
Besides, I don’t know what is your master mix components; Formamide is better than DMSO (to denature target sequences and primers).
I agree with the others. You can do a gradient PCR or PCR optimization considering each component
Missing data: size standard, information about the primers and expected product, origin and kind of template, PCR conditions.
There are a lot of possibilites. At least one of the PCR primers could bind at two sites in combination with repeated DNA regions, cyclic DNA, ... or ...
Hello Shahina
A PCR based gel does not look like this. First and foremost your DNA concentration is too high. As mentioned by Praveesh use 1 -10 ng for genomic DNA and 1-10 pg for plasmid DNA. You have to relook at your primers as you are getting multiple bands. Use gradient PCR to standardize your annealing temperature. Do not use high concentration of primers as you may get primer dimers in your gel. Lastly, use 1% DMSO in your master mix when you are fine tuning your PCR for better results.
May be problem with the primer that you are using. Cross check your primer once.
Cross Check your primer.
Change annealing temperature
Try nested PCR or Gradient PCR
you do the reaction with no template negative control.
Multiple bands could be from contamination, check purity of DNA.
Increase annealing time if the non-specific products are shorter than your target. If they are longer than you target, reduce the annealing time.
or redesign primer and also reduce the primer quantity.
Primers aren't so specific. Try to slow down annealing temperature to get more specific product and also do not exceed the quantity of sample (more primers and less sample get pcr reaction more specific). If this doesn't work, re-design one of the primers.
It seems that the primers are not specific. Please check the primers again.
The cause of the problem could be any of the following factors:
1: The primers are not specific enough for a particular DNA template
2: Annealing temperature is not optimal
3: Contamination
4: Overabundance of primers or overabundance of the template may be the cause.
5: Maybe MgCl2 concentration is not optimal or buffer is not optimal
Steps you can follow to get a clear band:
-Some bands are single and some are having multiple non-specific bands so there might be chances of contamination in your samples.
- Double check your primer because I have gone through the same problem and by a new stock my problem has been resolved.
- Check the concentration of gel buffer and running buffer.
- Try a gradient PCR or Touch-down PCR because annealing temperature can be the reason behind multiple bands.
Shahina Akter If you still have the problem, you can change PCR kit (different DNA polymerase) and reduce time for DNA synthesis. Next time, please, provide PCR protocol details. It would help to advice something more certain.
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