Hello, every one
if i have the following result how can get rid off nonspecific band if you know it is appear only with 2 tested sample as in lane 3,4 of my cDNA , but not appear with control (+) as in lane 2 , also no non specific band in my CDNA IN lane NTC dose not show up in contamination .
Thanks
Are you sure that lane 3 and 4 have no contamination with gDNA or something else? Did you run all of your different cDNAs in same PCR? I'm just wondering because the other fragments look quite fine.
I could also imagine that it could be a contamination with gDNA. Are your primers designed in a way that they cover two exons?
You also can observe unspecific products when the concentration of template is too low (e.g. not yet upregulated after short treatment time). So you could try using more cDNA for the PCR.
If concentration and/or a contamination are not a problem you can try to increase specificity by using DMSO or Betaine.
Good luck!
Daniel
I would add: try a higher reverse transcription temperature. Up to 60ºC, results can get much better (obviously not always), specially on GC rich and/or palindromic sequences.
I would first check what product size you would expect if your primers are amplifying genomic DNA. If it is matching the bands you see in Lane 3 & 4, then this is very likely to be gDNA contamination. So, unfortunately, you will have to either treat you RNA with Dnase again and make new cDNA or start again with extracting RNA. Good luck!
Hi Zeinab,
1. This is your RT-PCR result.
2. It looks like that this specific gene starts to express (mRNA) after 4-hour whatever treatment (faint band was observed at 4-hr point).
3. Why didn't you include a 0-hr treatment lane?
4. How do you know that the 'larger band' in lanes 3 and 4 are amplicons resulting from 'non-specific' amplification? Can it be, for example, a gene X which is related to the lower-band gene (and are gene family)? Is it possible that while the lower-band gene is not expressed (lanes 3 and 4), gene X expressed? Can the faint intensity of the larger PCR band is due to less conserved DNA sequences between these two genes?
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