I'm using the gene specific primer for screening the plant DNA samples. At the specific annealing temp (55) a 500 bp amplicon is getting amplified but there are significant non-specific bands too. I tried using different annealing temp (50 to 60 degree Celsius), diluting the samples (1:10, 1:5), changing the MgCl2 concentration but in the result either I'm getting non-specific band in addition to specific one or not getting any amplification (not even specific band).
Could you write your PCR conditions?
You can use DMSO (0.5ul/25 ul rxn) to reduce/ eleminate nonspecific band. But when you are using it, you should increase enzyme amount by %50.
Also you can try using BSA. From 10mg/ml stock, you can put 1-2 ul to 25ul rxn. But in two methods, you should use at least 100-200ng DNA.
Also with the receipt of your enzyme, you can try using touchdown PCR. 55'C annealing temp is low so it is very easy to get nonspecific bands. Start your PCR reaction with 62 degrees for 10 cycles than reduce for 0.4 degrees in each step for 20 cycles.
Could you write your PCR conditions?
You can use DMSO (0.5ul/25 ul rxn) to reduce/ eleminate nonspecific band. But when you are using it, you should increase enzyme amount by %50.
Also you can try using BSA. From 10mg/ml stock, you can put 1-2 ul to 25ul rxn. But in two methods, you should use at least 100-200ng DNA.
Also with the receipt of your enzyme, you can try using touchdown PCR. 55'C annealing temp is low so it is very easy to get nonspecific bands. Start your PCR reaction with 62 degrees for 10 cycles than reduce for 0.4 degrees in each step for 20 cycles.
You could start thinking that they could be specific targets.
My suggestion is to check primer sequences for specificity with Primer-BLAST.
Another option is a lenght polymorphism of the target sequence. You may purify each band for sequencing.
There are different ways to decrease of unspecific products of PCR
1. Using DMSO (BSA and Betaine is possible to use too)
2. Try to use another primers to the region of your interest (also check primer specifity)
3. Use high annealing temperature (as high as possible for your primers)
4. Be sure that you haven't contamination your DNA samples by nucleases and DNA is not degraded.
At least if you will not be able to aboid unspecific product formation you can separate all obtained products in agarose gel by electrophoresis. After this you can extract DNA product you interested in by the lenth. But all depends on how do you plane to use obtained DNA in your further research.
Best wishes
First: check if the the annealing temperature (Ta) you used is the right one, in fact Ta chosen for PCR relies directly on length and composition of the primers. In general, you should use an annealing temperature about 5°C below the Tm of your primers. The optimal annealing temperature (Ta Opt) for any given primer pair on a particular target can be calculated as:
Ta Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of product) - 25
where Tm of primer is the melting temperature of the less stable primer-template pair, and Tm of product is the melting temperature of the PCR product.
Optimal annealing temperatures will result in the highest product yield with the correct amplicon.
Second: which polymerase are you using?
DMSO will be the best choice....Still if it is giving more nonspecific product, change the primers rather than changing and seeing each and every component....
I agree to change primers, which is Tm >60 C, then try again and suggested the annealing temperatures also > 60 C. In addition, optimal decrease DNA polymerare concentration may improvement the condition. Good Luck~~
Try doing a nested PCR.
Depending on what you're working with, it could be splice variants, in which case you won't be able to get rid of them.
check primer specift.check primers on agarose gel for any degradations.reduce enz concentration.reduce anneal time.increase anneal temp up to see no band.reduce extention time.add enhancers.finally u can cut the correct band and purify it using gel extraction kit.and then reamplify the target
Amna Abdalla Mohammed Khalid Deniz Kom dear seniors, Hi, I also have the problem, but I 1st got very specific band (270bp) without any other band in a gradient at 62 (the highest with good result), and then I had to purify my pcr product for sequencing with ethanol and sodium acetate. few days later, when I run my reaction at 62c, it gave a second non specific band (below 100bp), I have run it then on three different thermocyclers but that extra band didn't go, what do you suggest could have caused this problem? thanking you
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