melting curve qrt-pcr - please,look at this picture! (Aug/27/2005 )
I need some advice!
Look at this melting curve!
I am new in Real Time PCR using SybrGreen!
I perform QRT-PCR on 7900HT apparatus with 384 plate, 15 microliter reaction. The plate has been centrifuged for 5 min. I use SybrGreen Mix from Abgene. This mastermix works very OK ( was recommended by another colleages) Standard curve of my assay seems to be not affected. Agarosa gel of this reaction shows a specific product and no primer dimer! The melting curves of the another 5 assays , which I try to establish, show very similar patterns of melting curves. The products are between 150 and 250 bp.
The attached picture is a melting curve from GAPGH, which has been used ( and proofed ) in SybrGreen assays by another labarotories.
I already called the AppliedBiosystems custome sevices, and they said to me, that they have never seen such melting curve before! Nice enigma!
I share the 7900HT cycler with a different another laboratories. And SybrGeen assays seems to be the very trivial thing in their hands, perfect melting curves. Such a melting curve creature seems to be my speciality! What is the systematic arror? Any idea?
Thanks a lot!
That is one totally wierd disociation curve.
I have never seen anything like that either.
wow... thats very pretty.
Silly suggestion - did you look at your reactions carefully - ie you didnt have any pen markings on the tubes or floating bits on the reactions???
Im not intelligent enough to come up with other suggestions!
Sometimes when the amplicon is long enough or it has regions of high differences in GC content, the melting curve tends to have strange shapes. Although I have never had a curve like that something similar happened to me with sybergreen. In my experience I couldnt find a solution and I guees it doesn't exists because it is not a problem of PCR but it's from the amplification sequence itself. So you should design new primers
Is this an earth quake plot..... ? ? !
By the way yu have a nice and uniform curve at 85-90oC. I think that represent your specific PCR product.
I am wondering why you got an earth quake like disociation curve, yet you don't see any primer dimer? Do you see any smear on your gel?
There are two way to get rid of. 1) you carry out a PCR purification first, mix your eluted PCR product with diluted SYBR Green (Cambrex has pure SYBR Green in solution form) and run melting curve again. 2) you may run your melting curve starting from 80oC all the way to 95oC. So that you will only see the peak of your specific product.