contamination in my real-time negative control

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help SA
help SA's picture
contamination in my real-time negative control

Hi

I have run a real-time PCR reaction with SYBR and included the following controls

1. Positive control: all the reactions mix with DNA from a target organism (V. cholerae)
2. Negative control: all the reactions mix with DNA from non-target organism (E. coli)
3. No template control: all the rreaction mix without DNA

I constantly get the same melt peaks as the positive in my negative control. I have also run gels and get the same band as my positive contro in my negative.

I have used this primers before and tested their specificity against different organisms including E. coli and they showed to be specific. I have tryed changing everything, meaning new primers, muster mix, water, wiped the bench with bleach but I still get contamination

Pls advise.

Ivan Delgado
Ivan Delgado's picture
Hi SA,

Hi SA,

From what I gather you are having problems with positive signals in your negative control. That is different from an assay being specific. Have you ever run a qPCR assay with this set of primers and gotten no signal in your negative control while having a strong signal in your positive control?

If you have cleaned everything up, used new reagents and primers, and you are still getting a signal in your negative control, then the issue is with your assay/primers. My recommendation would be to re-design the assay. 

help SA
help SA's picture
Hi Ivan

Hi Ivan

Thanks for the reply. I dont think the primers are a problem. I have used the same set of primers before in a conventional PCR and they worked fine. Now I want to convert this assay into a real-time PCR assay. I think my contamination is from the DNA extraction lab. I have just cleaned the DNA extraction lab now. I will then extract DNA from E. coli and see if it will be amplified. If it is amplified, i will  hav to consider re-designing the primers

Ivan Delgado
Ivan Delgado's picture
Just keep in mind that

Just keep in mind that standard PCR assays do not necessarily work as qPCR assays. For one thing qPCR assays work best when they generate small amplicons (no more than 100 bp to 200 bp).

Good luck.