Can these dissociation curves be used? - (Sep/06/2007 )
Hello,every one! I am new in Real time PCR.
Recently i met with some problems in my melting curves.There are always bimodal peaks or shoulder in the curves. But I run these problematic samples out on a gel and found that thest products were specific,there are no other products on the gel. So i am very confused about the curves .Can you give me some help?
this graph is the housekeeping gene GAPDH .I compare this graph with another primer pair GAPDH bought from toyobo company and found that the Cts of these two pairs from the same sample were nearly identical.I also run the products on the gel and found no other products. How can I explain this phenomenon?
these two cuves(blue and red) are the same primer pair and the products are specific confirmed by the gel. But how to explain the shoulder on the blue curve?
I am having similar problems with some primer sets.....I do not think those melting curves would be acceptable to use
I am still confused about the correct shape of the dissociation curve with single peak. I upload a picture which is from the stratagene company containing the dissociation curves. The notes beside the example 2 said the yellow curve in the presence of high template concentrations showed a high specific curve. But there is a distinct shoulder before the peak. I think my blue one curve in that picture also seems like this. Is that wrong?
It is difficult to see these pictures - but personally I have discarded primers which give a shoulder like that if further optimisation can not remove it. I have not come across any published papers in my field which display such curves
ok,I will consider this picture unacceptable.
But i also have some other problems to consult:
1. at first, I bought GAPDH primer from toyobo company and the melting curve is very good.But after one month when I did the real time pcr with the same primer another time, all melting curve has a shoulder before the peak. I run the products of 140bp out on 3% gel for 47 mins and found them specific. The only difference in the conditions is that I take two steps in the amplifications the second time. I was very confused about such big difference and do not know how to solve it? Can you give me some advices?
Hi, I've just started with qPCR too, so I'm not that experienced, but I've been to a meeting once and Thomas Kaiser from Corbett said it's alway good to keep the times for annealing and elongation as short as possible to avoid by-products. Maybe it's worth a try.
Good luck!
But i also have some other problems to consult:
1. at first, I bought GAPDH primer from toyobo company and the melting curve is very good.But after one month when I did the real time pcr with the same primer another time, all melting curve has a shoulder before the peak. I run the products of 140bp out on 3% gel for 47 mins and found them specific. The only difference in the conditions is that I take two steps in the amplifications the second time. I was very confused about such big difference and do not know how to solve it? Can you give me some advices?
The figure legend you posted in your third post indicates that the starting concentration is important.
I have found this to be the case in my standard curves - The higher concentrations give nicer peaks. What sorts of Cts are you seeing?
Another possibility is that you might have two different alleles of the same gene in your reaction? Is that possible? I will sometimes find that different alleles will have different Tms (depending on GC content etc).
But first I would look at the Ct, and if it is higher than about 28 - 30 or so I would think that this might be why you are getting a shoulder.
(Remember that SYBR recognises double stranded DNA, and so anything that is promoting replication in your mix - even if it is low-level non-specific replication between DNA junk in the sample, will allow SYBR to bind. The PCR product will ultimately win out because of the molarity of primers, but any sort of background could be due to non specific priming/replication of non-target DNA. Try raising your anneal temp, or try Taqman.)