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melting temperature in real time pcr


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#1 miss-pcr

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Posted 23 November 2010 - 12:32 AM

why melting temperature gives some times more than one reading in real time pcr results.
thank you

#2 HomeBrew

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Posted 23 November 2010 - 03:31 AM

Do you mean multiple melting temperatures in a single experiment, or variation in melting temperature between different replicates of the same experiment? Melting temperature is determined by the base composition of the fragment being melted -- if you have multiple fragments of the same size but of different base composition, they will melt at different temperatures.

#3 ivanbio

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Posted 23 November 2010 - 09:40 AM

There are a number of reasons why you may get multiple readings for melting temperature. Here are some:

1. The second peak could be primer dimers
2. Your PCR amplicon is long and/or complex enough that more than one melting region is present. Read about HRM for more details about this
3. As HomeBrew stated, if you have multiple amplicons with different DNA sequences, they may likely melt at different temperatures
4. You have enough DNA contamination that you are picking up other melting peaks

The bottom line: if you should not be getting multiple melting temperatures (i.e. this was not expected), you may need to optimize the assay or re-design it.

Ivan
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#4 miss-pcr

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Posted 23 November 2010 - 10:36 PM

Do you mean multiple melting temperatures in a single experiment, or variation in melting temperature between different replicates of the same experiment? Melting temperature is determined by the base composition of the fragment being melted -- if you have multiple fragments of the same size but of different base composition, they will melt at different temperatures.

Thank you very much "Homebrew" for your reply...what I mean is some times I get more than one Tm reading for one peak

#5 miss-pcr

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Posted 23 November 2010 - 10:38 PM

There are a number of reasons why you may get multiple readings for melting temperature. Here are some:

1. The second peak could be primer dimers
2. Your PCR amplicon is long and/or complex enough that more than one melting region is present. Read about HRM for more details about this
3. As HomeBrew stated, if you have multiple amplicons with different DNA sequences, they may likely melt at different temperatures
4. You have enough DNA contamination that you are picking up other melting peaks

The bottom line: if you should not be getting multiple melting temperatures (i.e. this was not expected), you may need to optimize the assay or re-design it.

Thank you very much "ivanbio" for your reply...what I mean is some times I get more than one Tm reading for one peak

#6 miss-pcr

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Posted 23 November 2010 - 11:03 PM

Thank you very much "Homebrew" and "ivanbio" for your reply...what I mean is some times I get more than one Tm reading for one peak for example one peak gives 85 the other gives 92,and what is the relation between these readings and the bp size of the DNA product.

Thank you very much for your cooperation




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