I'm trying to quantify bacterial genomic DNA from stool samples absolutely with an already published Assay (http://gut.bmj.com/content/53/4/523) and having some trouble since the amount of detectable DNA is very low.
My standard curve works fine over at least 5 logs (Cq 17.5-31.5) with an overall efficiency of around 95 %.
The bacterial DNA for the standard is from one species within this genus and was extracted with a commercial kit.
The primers capture my standard species as well as other species of this bacterial family.
Primer concentration is 500 nM each primer and I'm using Power SYBR Master Mix from ABI in a 25 µL reaction.
My stool samples come from patients with rather nasty bowel diseases and hence even after the DNA extraction (the method combines heating, mechanical and enzymatic steps) the samples tend to be somewhat dirty.
The ratio for 260/280 is usually > 1.8 but 260/230 is poor (between 0.7 and 1.8 depending on the sample).
I normally have to dilute my samples to at least 1:10 prior to using them to get no inhibition.
My actual problem is the following:
the Applied Biosystems 7500 Software flags most of my samples with 'multiple Tm peaks' (even the last two of my standards with Cq > 34)
Can anybody explain why? I can see no peak at the given melting temperature and don't know what should be there anyways. Primer dimers should come at a higher melting temperature.
Attached there are 2 files of the derivative and normative melting curves for selected samples:
1 are my standards (Cq 17.5-31.5)
2 is my standard at Cq of 34.8
3 is my sample at Cq of 37.07, 37.27 & 37.68
(i've just selected one of three triplicates for the standard)
The last two pictures show the amplification and melting curve plot for all my samples, standards and no template controls (NTCs).
I know I shouldn't use my sample with a Cq of 37 anyways (at least I was told so because of the late cycle) but why do I get a MTP flag? The programme tells me that the 2nd melting peak is usually detected at 60 to 63 °C.
I haven't run an ethidium bromide gel yet.
Furthermore: the authors of the assay I'm using get a specific melting curve at 89 °C (see paper above) and they say that this is specific to the genus of the wanted bacteria.
My peaks come between 80 and 83 °C. Is this maybe due to different species (and hence different amplicon melting temperatures) that are captured?
Thanks alot for your answers.
Edited by Archie, 02 November 2011 - 03:37 AM.