I am amplifying gene (1488 bp) from RNA genome using specific primers and run in reverse transcription PCR(RT-PCR). The result, I am getting 2 bands my target band (1488bp) and extra band with size ~ 400bp)
To remove the extra band, I cut my target band from gel and purified my product by using Purification from gel -Agarose Gel DNA Extraction Kit. Then amplified PCR product, but I got the same result my target band (1488bp) and extra band with same size(400bp) .
I repeated this several time and every time found the same thing.
Tried using a different annealing Temp(high and low) , but the problem persisted.
i attached the pic of gel (lane 1 ladder, 2 and 3 lane PCR product )
I would really appreciate any advice on how to troubleshoot this issue.
It sounds like your long sequence has an internal site that the primers can bind to so your 1488 band is 1-1488 but the primer 1-20 also exists at 1088-1108 so you can amplify both products and the smaller product will amplify better because it melts and extends more efficiently, Check your amplimer for both forward and reverse primer sequences
2 possible reasons I can think of:
A) As previously mentioned you have and internal primer binding site. You must redesign primers to get a more specific amplicon. I'd suggest design a primer pair with a Tm near/equal to 72 celsius and perform a two step or touchdown PCR.
B) You have an internal region where the polymerase is falling of the replication cycle. Check for stretches of AT or low complexities or pseudo terminators, in that case you will always get the two bands and you should amplify the two regions using separate but overlapping primers and then do cloning using LIC via Overlap-Extension PCR or Gibson/Hi-Fi builder protocols.
If I understand you correctly, you are conducting RT-PCR for expression analysis of a gene of interest. The second, smaller band may simply be a splice variant. I would suggest cutting out both bands, purifying them and directly sequencing them; or you clone both bands and then sequence the plasmids. Sequence analysis will show what the second smaller band is and whether it is indeed a splice variant.
Even if you cut out your band of interest from the gel and purify it, there will always be some traces of other amplification products retained, which reamplify as well.
I had sequencing the smaller band, I found that it shared similarities with my target gene, but it was only a part of it. I redesigned the primer(make it longer and chang CG%), but unfortunately, I still obtained two bands - one for my desired gene and the other for the undesired one. Can you please help me to figure out how to amplify only my target gene? I have to isolate whole gene for next step (gene cloning and expression )
I utilize the QIAGEN onestep RT-PCR kit for my research involving the isolation of my target gene from an RNA genome, which is subsequently cloned and expressed.
You could either amplify,run the gel and gel purify the long band for your next stage or add some bases to the outer primers so you can run the pcr at a much higher annealing temperature and hope the internal primer does not anneal at this temperature ( check the sequences) or possibly run the pcr with DMSO at 2%, 4%, 6% and 8% and hope that the pcr only works with the correct primers at some percantage of dmso
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