After many trial and errors I got a band in my PCR amplification. I have used taq, DNTP mix from the same company (Termo scientific). I think it could have helped me. But now the problem is the bands looks strange. I got primer dimer and also a band above the dimer. Is it my band of interest? I am expecting my band with a size of 200-300 bp. I am attaching the gel pic also with this. In the pic there are not much separation between the dimer and band. Please help.
Hi,
You should use a DNA ladder with small fragments to estimate the size of your PCR product. It is almost impossible to know if it is your product of interest without a ladder or at least further information about run time, voltage and gel size. The size of your fragment might be in the range of your expectation. However, it could be just 100bp as well. An increased agarose concentration (e.g. 3%) will enhance the separation of small DNA molecules by gel electrophoresis.
Best,
Boas
Hi,
You should use a DNA ladder with small fragments to estimate the size of your PCR product. It is almost impossible to know if it is your product of interest without a ladder or at least further information about run time, voltage and gel size. The size of your fragment might be in the range of your expectation. However, it could be just 100bp as well. An increased agarose concentration (e.g. 3%) will enhance the separation of small DNA molecules by gel electrophoresis.
Best,
Boas
Hi,
I agree with boas suggestion. Without a ladder you cannot differentiate your DNA. If you want to know what the other band might be increase your agarose gel concentration.
The run time was approximately one hour with a voltage of 100 v. The bands were run on a gel size of 30 ml. about10 centi metres.
Hi Keerthy,
to answer whether the bands contain your sequence of interest you should a) use are more concentrated gel with an appropiate DNA ladder (as recommended by Boas and Balaraman) and b) cut out the band that you think might be the one, clone it into a simple vector like for example pGEM-T and let it get sequenced. This way you be sure that it is the right band.
Hi
You need to load a ladder to be able to judge if it is your band or not
Hello,
As many suggested, use a DNA ladder first and also keep a Non template control (NTC) i.e. mastermix without cDNA along with sample which will help you to troubleshoot your problem. For better resolution of use higher percentage of gel as suggested by Boas.
Hope this helps.
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