We are running qPCR for the quantification of HCV RNA. For the past two weeks, we seem to get amplification curves and Cts with:
1. Negative samples
2. Water + Mastermix + primers+ probe
3. Mastermix+primers and probe.
When we run a gel for the PCR product we get a 250bp band for (1-3) which is the size of our amplified region! If we use a different set primers, that work on a different region we get no Cts for (1-3). We think we have contamination by our PCR product. In attempt to get rid of it, we have tried :
1. Using new reagents ( RNA extraction kit, mastermix, primers, probes and water)
2. Bleaching our pippettes and work space
3. Using another safety cabinet for RNA extraction and PCR preparation.
4. Using another PCR machine.
5. Not using a probe and running conventional PCR on samples (1-3) detailed above but without probe.
All of that did not help! Any idea on how to get rid of it.
Thank you
Hi, I think you have taken many measures to avoid contamination and to trace back the problems. But all your efforts didn't work unfortunately and sorry to hear that.
Another possibility is maybe your RNA sample has problem, eg: contaminated./degraded You may try to get new samples and re-extract the RNA by using new reagents and repeat your step 1-5. Try to think back whether your RNA source has problem, storage problem etc?
In many circumstances, contamination and not getting desired results on molecular work can be really frustrating and un-explainable. This might sound "non-scientific", but sometimes I will just pray hard to hope that everything goes smooth whenever the results keep disappointing me.
Hello Salma,
Really impressed by the way you've worked to get rid of the contamination. Looks like there is nothing else you could do! But have you tried a new pair of hands?? There are chances that whenever we do the same experiment again & again we might miss something. Ask one of your colleagues/fellow researcher to perform the experiment for you. Then look at the results. A new set of eyes can actually help you find out what you missed!
Best!
Sounds like you have done everything to solve the problem. I used to have contamination problems too. So before starting every PCR i always clean my pipettes and first i prepare the master mix + water and primers. Then I put furst the water for the blank and distribute my cDNAs to reduce any risk that the water may be contaminated with cDNAs...and then when i distribute the master mix i start always with the blank and then continue with the cDNAs. So if i use a pipette for cDNA i prefer to clean it first before using it for water or the other reagents. It is even better if you have a pipette exclusive for cDNAs i think. Maybe this can help, all the best.
I think it could be primer dimers. When you used a different set of primer, the problem seemed gone. Have you tried increasing the Tm? Or sequencing your PCR product?
Use bleach and/or UV irradiation and continue working in a HEPA filtered hood
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