Hi everyone, so I have been running some assays in qpcr(presence/absence experiments).
My experiment is on detection of human malaria parasites.
My positive controls show as present and usually above the target threshold.
However my samples are usually below the target threshold although they yield some curves.
The positive controls have Ct's around 20 and the samples Ct's range from 26 to 30. I use 5ul of DNA for this assay.
I ALSO USE THE ABI 7500 FAST REAL TIME MACHINE.
How do I fix this?
PLEASE NOTE: I have attached some pictures of the results
Thanks.
Hi Irene-Love Amoah,
Having curves for the target gene means that there is an amplification (positive reactions) but we have to validate the result before any interpretation:
First, to discard any possible contamination, did you put negative controls? Are they negative?
Second is your PCR set (primers+probe) specific to the target sequence?
Hi Aissa Saida, yes I did put negative controls. The negative control curve was flat to a point but begun to rise a little at later Clcycles.
They were below the threshold though so I am considering it as not very significant. What do you think?
Also my primers and probes are designed specifically for the targets
My concern is if there is amplification why do the results read as target 'unconfirmed' ? and "present" for only the positive controls?
Irene-Love Amoah Your samples have curves looking like obtained with significant inhibition of PCR. You can try two-three different protocols/kits for DNA extraction.
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays