I am a new member and this is my first post and I am sorry for a long post.
I need to amplify a particular DNA from a mixture of two. My target DNA in the mixture could be from 0.1% to 10% only. I am trying to develop qPCR for this. Our designed primers are working well and up to the expectations/requirement in terms of sensitivity and specificity in end point PCR. In end point PCR, we have eliminated the primer dimmer formation by optimising the Tm (with little compromise on the amount of end product).
However the same is not true in either SYBR green or in taqman PCR. In SYBR based there is lot of background noice (non-specific fluorescence) and upon agarose gel analysis the primer dimmers re-appeared. Therefore we designed taqman probe/s.
In the taqman probe assay....
I am trying to standardise the assay first by using pure extraction of my target DNA (instead from the mixtures) with range of concentrations from 0.1ng/ul to 20ng/ul (I am making dilutions beginning with 20ng/ul down to 0.1 ng/ul).
The problems are…….
1. No sensitivity (in the lower range concentrations i.e., <5ng/ul no amplifications seen).
2. No identical results in the duplicates or triplicates especially in the lower range (<5ng/ul).
3. Even at 20ng/ul concentrations also the Ct values are above 30.
We want to bring the Ct values to at least 26, also increase the sensitivity levels of detection.
We have tried to optimised the primer concentrations with primer matrix and it really did not have any effect.
I have tried 1. Invitrogen SYBR green qPCR superemix-UDG
2. Abgene’s ABsolute Fast QPCR Low ROX Mix
3. ABsolute QPCR Low ROX Mix (non Fast enzyme).
The later two are with taqman probe based assay. We have optimized the cycling programme for taqman probe based assay.
The problem in the replicates is not due to pippeting errors and we ruled out the possibility and also for the fact that I work from the master mixes. One possibility that while making dilutions from 20ng to 0.1ng insufficient dissolving/suspension of DNA as I only mix by pippeting up and down. As a solution for this problem, each diluted sample has been vortexd & centrifuged, left overnight at room temperature and were nanodrop quantified twice (once immediately after vortex-centrifuging and second time is after overnight incubation at room temperature). The measurements were taken in duplicates. There are two problems with this
(i). Large dynamic range is 2-3700 ng/ul (dsDNA) on a single sample and so I can’t quantify <2ng/ul.
(ii) The results of quantification before and after over night incubation are not reproducible.
Any suggestions 1) to get identical results in replicates. 2) To increase sensitivity levels of detection down to 0.1ng/ul DNA. 3) To get Ct values of around 26.