My lab is trying to do RT-PCR, by converting RNA to cDNA and then using the standard 16S bacterial primers and PCR conditions on it. Unfortunately all we get are giant streaks with no discernible bands. With other primers, we have normal bands. What could explain this? Any suggestions?
That sounds like RNA degradation to me
Google how to solve as for me to go over it all in one answer would be too much
If you want to come back to me on specific things you read drop me a line
In this type of question including a picture of the gel image is often helpful Matan Shelomi
What did your PCR controls look like? That can help narrow down the possible list of issues.
Giant streaks on Gell, Are you using specific primers? Also, check the Gell whether it is dirty or not? Put appropriate markers in the Gell,
It does not look like too much dna template as the other amplifications look clean of smears so I think that you have post pcr contamination. One of your reagents/pipettes/plasticware/working area is contaminated with amplimer. The amplimer re amplifies hugely and very efficiently to produce so much amplimer that it self anneals and concatenates to form long smears. What do your no template control samples look like. They should be clean but and amplification of water in the NTC means that you have contamination
Hi Matan Shelomi , with my personal experience I can say that this happens when too much RNA is used for making cDNA. Although I have a question, the bands shown with other primer pairs, are these primers also specific to 16s rRNA or some mRNA? As mRNA is very less in concentration relative to rRNA.
Thanks everyone for your answers!
In our case, the problem was insufficient extension time during RT, so our full-length 16S primers could not work, but primers for smaller-length 16S sections worked fine.
- Badges
- Science method