real-time PCR - Multiple melting curve peaks (Aug/23/2005 )

search for a software called FastpCR, it is a freeware.
Plus. I have just done a quick search about those later sequences of yours, the software indicates no primers or hairpin may form.

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thankyou!!!!!!!

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looking at your signal level , it looks as if your target didn't get amp. did you check you product on a gel to verify you have one single PCR product or a pcr propduct?

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Yes the signal level is quiet lousy. Have you made a blank/not template control just with probes? How does the curve look like? Just check your aplification first.
With population I mean that there are two probes; simple example: xxxagttxxx and xxxacttxxx and therefor there is double peak just even at the wildtype.
The secondary structure might be a problem as well. What are the melting points of the probes? the first peak might be dued to this.

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Hi,
yes all checked on gel - 1 PCR product and i digeted it and it is definately the right PCR product.

nice to know I did something right!

Thanks
edwina x

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I tried making a non template control - but I constantly got contamination - which I know was a valid PCR product as I gel viewed it and digested it to get heterozygote DNA - so hence in that sample even with the contamination it should only have 2 peaks - the mutant from the original sample and the wild-type DNA from the contamination.

I didnt run a sample with just the probes alone. I will give it a go (although will have to go in my MSc as a further work thing as its due next week)

The probe melting temp were;
probe 1; 61C
PROBE 2; 71C

I did wonder whether this would be a problem...do you think it may have caused an extra peak in the graph?


Thanks so much for your help/suggestions - supervisor is as much as a chocolate teapot! - asked me yesterday what a primer was!

Thanks
edwina

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[quote=edwina,Aug 25 2005, 11:01 AM]
[quote=nabla,Aug 25 2005, 07:49 AM]Yes the signal level is quiet lousy. Have you made a blank/not template control just with probes? How does the curve look like? Just check your aplification first.
With population I mean that there are two probes; simple example: xxxagttxxx and xxxacttxxx and therefor there is double peak just even at the wildtype.
The secondary structure might be a problem as well. What are the melting points of the probes? the first peak might be dued to this.

[/quote]

I see what you mean about the probe populations now... maybe this is a problem... but I had the probes made and checked with HPLC by a commercial company.. so I would be surprised if they werent made well.

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Contamination is a problem you will have to solve! Practically you can not use you data then?
Just at a second look at your melting curve. Don´t you need the first negative derivative to present the graph?
Mistakes can always happen: We had the problem that the probes were without phosphate thus the experiment didn´t work at all, because the probes were elongated. And last time I had a primer-population in one test!
Greetz nabla

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No practially I cant - but I can make some deduction as i know what the negative contamination genotype is.
Im pretty certain the graph is the negative derivative - ie -df/sddt vs temperature.

As it is my practical time is over for my project so I cant include any more results in my write up- which is a shame.
I amy however persue it anyway as my hospital laboratory need to set up this genotyping method either using this real-time or RFLP. And I do think - (once it works) - the real-time has many advantages.

Thanks so much for your comments.
This forums been great - at least I can finally speak to someone with experience - or at leaset knows what I am talking about! My boss/supervisor is totally ignorant about all molecular biology, including PCR!

Have a great weekend
Edwina
x

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