Even though we had an expected product size in SSR amplification sometimes more number of bands are visible in agarose gel electrophoresis;How can we avoid this?
Hi!
First of all upload or send your gel photograph along with PCR conditions/componenst to me then I advise.
Many reasons for non specific amplifications. How many times you have done the PCR? With how many samples! You have to check right from your PCR components, hope they all are fine and not contaminated. You have to check your Ta. Are you following Hot-start? Too much of MgCl2 also cause problem. List your components how much you are taking per reaction. Without seeing gel pic. I can not tell guess anything. If you search in RG you come across so many question answers of same type.
Hi,
Jetty Ramadevi has rightly pointed out. There are many factors.. that needs to be considered. First thing is you should check the Tm of primers, run a gradient PCR to optimize the annealing temperature. Also, check for MgCl2, it should not be too high. Its always better to go for a hot start one that reduces non-specificity. If everything is fine in your reaction set up and also it is reproducible after multiple repeats, then you can think about multiple alleles or heterozygosity. But before concluding any such thing you must have to be very sure about correctness of your experiment handling.
If you are sure for experimental conditions suggested by Dr. Pandit and Dr. Ramadevi, It means that your primers targeted more than one loci due to paralog loci . In such a case you can score PCR products desperately for mapping such as SSR1a and SSR1b.
Non specific amplification for SSR loci can be due to various reasons: Primer designing, annealing temperature, amount of Genomics DNA you are using. If you are sure of primers designed and other variables in PCR reaction, try to reduce the number of cycles for PCR reaction, It generally varies from 25 to 35.
Apart from the above-mentioned suggestions, multiple bands can be obtained when you design your primers from such genes or ESTs whose copy numbers are multiple in that particular species or organism.
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