Hello everybody,
I was wondering to know how we can remove the primer dimer from PCR product in a way that, the quality and sharpness of the desired band remains unchanged.
Hi Paria, you'll probably have to do a bit of troubleshooting with your PCR. Try lowering/raising your annealing temperature, try using less DNA, try changing the volumes of reagents in your pcr master mix, and maybe try extracting new DNA, if possible, in case your current working stock is beginning to degrade.
Hi Paria, you'll probably have to do a bit of troubleshooting with your PCR. Try lowering/raising your annealing temperature, try using less DNA, try changing the volumes of reagents in your pcr master mix, and maybe try extracting new DNA, if possible, in case your current working stock is beginning to degrade.
If you want to get rid of the band forming in the PCR, then try increasing the Mg2+ a bit (1mM at a time) and increasing the DNA template a little (but not above 100ng max for a whole vertebrate genome or proportionately less for smaller genomes in a 10ul PCR). To get rid of most of a primer dimer band, post-PCR, you can use size selection with one of several kits so long as your product is above about 100bp. For example the Qiagen PCR clean up kit does this. But it is not cheap!
the best way not to get PD in a pcr reaction is to design good primers which do not dimerise. There are online programs like primer3 which will help. Failing that use less primer ( start with 1/4 the amount you are using) and a hot start enzyme. If cost is an issue make the pcr mix without enzyme,heat it to 80c then add the normal taq enzyme to the hot mixture and start cycling without letting the mixture cool down.. Run a temperature gradient to find a high annealing temperature for your primer set which should cut down the amount of primer dimer
Add additive such as DMSO or Betaine, which lower the melting temperature of the primers, avoid PD and disrupt secondary structure formation. Sometimes it is impossible to design primers that form primer dimers or secondary structures like in case of high GC content templates, where the only option is to design optimum size primer and use such additives. I amplified genes with 70-73% GC content with a commercial GC enhancer.
Silica column based PCR cleanup kits are good enough for cleaning and allow elution in the desired concentration.
Sometimes dimers are unavoidable. Decreasing the amount of primers can sometimes help. Another method would be to let your samples run out longer on your agarose gel. That will give you more separation between your desired product and the dimers.
In addition to all the suggestions, increase your reaction volume. if you total PCR reaction is 20-25ul, scale up to 50ul.
Hi,
You should change your annealing temperature of thermal profile (raising the temp.). Simultaneously you have to increase the PCR reaction volume, lower the conc. of the primer and delayed the annealing and extension time in your PCR cycles. This may help you.
Agree with Ashoke Kumar Bishoyi, you may try to adjust your annealing temperature by running gradient PCR...
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