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Informations about melting temperature

Informations about melting temperature - Real-Time PCR and Quantitative PCR Forum

Informations about melting temperature - Real-Time PCR and Quantitative PCR Forum


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Old 02-17-2009, 09:54 AM
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Exclamation Informations about melting temperature



Hi all,
I'm trying to build an Internal amplification control to my real Time PCR adding a fragment of 50-70 bp to my PCR amplicon. The problem is when I check for the melting temperature (MT) of this internal control I found (with many oligo checking software) a lower MT than the amplicon. Does somebody know why?
I had alway belived the temperature melting amplicon's was directly proportional to the GC content and more general to the lenght of the fragment.
Many thanks in advances
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Old 02-17-2009, 11:58 AM
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Default Re: Informations about melting temperature

From my experience it is the GC content and the arrangement of GC nucleotides that play more important role than number of bp in amplicon.

One day I amplified two fragments - one 288pb and the other 487bp. I suspected that longer amplicon would have higher Tm, but it was the opposite, and the difference was huge - shorter amplicon had melting temp. at 89°C and longer at 80.5°C. This is due to CG content and arrangement. Long CG clusters significantly increase Tm of an amplicon.
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Old 02-20-2009, 02:58 PM
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Default Re: Informations about melting temperature

Hi Aga,
thank you very much for the answer, shall I ask You if You know any free software able to measure the Tm also for long oligo? Cause I suspect all' equations currently used are specific for short oilgo (less than 30 bp).
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Old 02-20-2009, 08:35 PM
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Default Re: Informations about melting temperature

OligoAnalyzer: [Only registered users see links. ]

Input your sequence - and click 'Analyze' - underneath you should receive not only melting temperature of your oligo, but also molecular weight, CG content, extinction coefficient etc.

The melting temp. is accurate for 8-60bp, but you can input longer sequences and it will be calculated anyway. In this case I don't know what's the accuracy. So try these two:

This one gives you Tm calculated using basic, salt adjusted and nearest neighbor methods: [Only registered users see links. ]

The next will give you even more info: [Only registered users see links. ] They also tell you what is the idea behind each Tm calculating method.

I checked them all - you can input longer oligo sequences.
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Old 02-26-2009, 11:30 AM
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Default Re: Informations about melting temperature

Hi Aga,
I known either oligoanalyzer than oligocalc, but I has never heard about the promega software. In my opinion the first is the best, cause the Tm predicted was the same I found out with my stepone plus from AB.
Thank you very much
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