Dear all,
These are data from gel base of cDNA sample for PCR with protocol attached on the figure. Band 5 is no reverse transcriptase enzyme during doing cDNA library used random primer. With gen A, band 5 is very clear, strong. Has any people got this problem? Why did it happen? How to solve this problem?
I have tried more than 5 genes: gene A, B, C, D, E (for example). Just only gene A is got this problem. I have changed other two different gene A primer sets to check again, but I also got the same problem.
Thank you for your help!
Mita
does gene A have a known pseudogene that is amplifying from gDNA perhaps?
Have you sequenced any of the amplimers of geneA to eliminate the possibility that they are all the wrong amplimer and the primers are just annealing in the wrong place and amplifying off gDNA from an unrelated sequence?
It might be worth amplifying gDNA with these primers to see if that is where the bands originate
does gene A have a known pseudogene that is amplifying from gDNA perhaps?
Have you sequenced any of the amplimers of geneA to eliminate the possibility that they are all the wrong amplimer and the primers are just annealing in the wrong place and amplifying off gDNA from an unrelated sequence?
It might be worth amplifying gDNA with these primers to see if that is where the bands originate
I would concure with Paul Rutland this is most likely due to genomic DNA. I would also recommend making sure that your primers for RNA span exons such that you should not amplify from the genomic DNA.
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