I have even tried different annealing temp but I am getting double bands in all the temperatures I have tried.
I suggest you sequence both fragments. That will give you an idea of what going on. If you are amplifying the CDS of a gene it is possible that it has a corresponding pseudogene somewhere in the genome. It happened accidentally to me.
Otherwise you have to play more with PCR conditions. Use less Mg, add betaine 1.5 M, etc…or even redesign better primers.
Do you have the expectant size? If so, cut out that band, sequence it and redesign primers. To cut out you can separate on polyacrilamide
Brijesh -- have you considered varying the magnesium concentrations or adding DMSO? I'd try 3% DMSO to start and see how that works. As above, you could gel-purify the band of the appropriate size and clone that one (I am presuming you're cloning, so the following goes out the window if you're simply looking to detect a gene) -- or simply put the PCR product into your cloning reaction and miniprep and screen the resultant colonies for your product of interest. This second idea is likely a better one if the product you want is more abundant or smaller than the other (unexpected) band.
The good news -- when cloning a gene, you only need to succeed once.
Check your primers first. You might have two sequences that are being used as targets (templates). I recommend running your PCR electronically at http://www.ncbi.nlm.nih.gov/projects/e-pcr/ . It could be that you have two sequences that are very homologous. If cloning and sequencing are not issues, I would sequence both. I have found sequences coding for isozymes this way. Some sequences are very similar and only differ in a few nucleotides. Some times the two bands are the results of non-specific primer binding but I would not discard the possibility of discovering a homologous sequence by accident.
I suggest you sequence both fragments. That will give you an idea of what going on. If you are amplifying the CDS of a gene it is possible that it has a corresponding pseudogene somewhere in the genome. It happened accidentally to me.
Otherwise you have to play more with PCR conditions. Use less Mg, add betaine 1.5 M, etc…or even redesign better primers.
Try raising the annealing temperature using a gradient, and/or optimisation using DMSO or MgCl2. Touchdown might be worth a try, too. If not, you may have to re-think your primer design. Good luck.
You may use touchdown thermal profile, from 65 to 55. Start with annealing 65 and decrease it1 C every cycle until you reach 55. Then do 25 cycles at 55.
Dear Brijesh : First of all as suggested by others let us know if you are getting your interest amplicon or not? if not then certainly you can change some of the parameters of PCR (like Mg conc, add DMSO , will reduce non-specificity , may go up with the tm etc ). If yes then simply elute it and sequence it, it is always better to design a primer from exon-exon junction and moreover checking it via Blast (so that you know it only amplify your target sequence or not). Hope it will help
Best
I made the experience that to low concentrations of target sequence in my reaction mix can cause my primers to form dimers when they have a tendency to do so. May be an increase in the sample volume or the concentration of your DNA solution can better your results.
What is your template? Do you amplifiy from plasmidor genomic DNA? If u apply plasmid DNA then digest plamid outside of GOI with restriction enzymes and re-do PCR (unwanted fragment cannot be amplified exponentially). Otherwise stick to comment 1 and 2 and simply extract DNA fragment from agarosegel, clone and sequence it. Generally it would be helpful if you provide more information on the template - which seems to be the crucial part. Good luck!
my template is genomic DNA and yes i got the right amplicon size but the problem is along with that another second band is also seen. I have tried annealing temp from 55 to 65 c but at all temp i got two bands.As suggested above to use DMSO, how much concentration is appropriate?
Ok then I would go for the strategy suggested by Abdelhalim Boukaba. I am not sure what you plan to do with the DNA fragment - however a simple agarosegel extraction followed by cloning and sequecing would be the next thing I would do. This will isolate and purify your fragment sufficiently and you can use it later for any other purpose.
Hi! Probably you have unespecific amplification. Are you using positive controls? You should improve your PCR conditions and temperature. Some strategies for reduce this kind of amplifications could be: increasing annealing temperature, incrising Mg, use less DNA... Use positive controls to optimize your PCR. You can also extract both bands from the agarose gel and purify them with a specific kit and then sequence.
What is exactly the Tm of your primers? And which algorithm you use to determine the Tm? Each software use different algorithms, so you estimate a Tm which is much more different than the real. I use Gradient PCR from 50-70 when I have a Tm around 58. With different concentrations of MgCl2 (1,5-2,5).
it is Nonspecific Bands or Primer-Dimers , try to visit this link
http://www.bio-rad.com/en-us/applications-technologies/pcr-troubleshooting?ID=LUSO3HC4S#gel2
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