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restriction sites to be included while calculating Tm?


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#1 balancer

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Posted 03 July 2010 - 05:38 AM

I have used softwares such as Pcr primer stats earlier for calculating Tm.
My present lab uses geneious software, where in they don't consider addition of restriction sites and extra bases while calculating Tm.
To come to think of it, its right as restriction sites and extra bases forms over hangs in first round of PCR.
So am not sure, which method to be followed as both methods give different tm values.
Kindly suggest the right way.

#2 Curtis

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Posted 03 July 2010 - 06:58 AM

Tm is calculated for the gene fragment. That's why they don't consider RE and overhangs. I remember I had the same question when I was learning this.

It is because in the first cycle your overhang and RE site don't bind to any complementary sequence at all. It is at the annealing step of 3rd cycle when the whole primer finds a full complementary sequence to bind. When your primers have over hang and RE site your first PCR product will be produced at the end of cycle 3. Draw it on a paper and you will understand.


I have used softwares such as Pcr primer stats earlier for calculating Tm.
My present lab uses geneious software, where in they don't consider addition of restriction sites and extra bases while calculating Tm.
To come to think of it, its right as restriction sites and extra bases forms over hangs in first round of PCR.
So am not sure, which method to be followed as both methods give different tm values.
Kindly suggest the right way.


Edited by Curtis, 03 July 2010 - 07:02 AM.


#3 balancer

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Posted 03 July 2010 - 09:45 AM

Tm is calculated for the gene fragment. That's why they don't consider RE and overhangs. I remember I had the same question when I was learning this.

It is because in the first cycle your overhang and RE site don't bind to any complementary sequence at all. It is at the annealing step of 3rd cycle when the whole primer finds a full complementary sequence to bind. When your primers have over hang and RE site your first PCR product will be produced at the end of cycle 3. Draw it on a paper and you will understand.


I have used softwares such as Pcr primer stats earlier for calculating Tm.
My present lab uses geneious software, where in they don't consider addition of restriction sites and extra bases while calculating Tm.
To come to think of it, its right as restriction sites and extra bases forms over hangs in first round of PCR.
So am not sure, which method to be followed as both methods give different tm values.
Kindly suggest the right way.



Thank you Curtis. How to go about setting up annealing temperature, as on the primer tube ordered from company has a different Tm?
I kept 5 degree below than what given on the tube. If it does not work, i set up a gradient. Please suggest.

#4 Curtis

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Posted 04 July 2010 - 02:56 AM

yeah, if you have enough sample then better run gradient. I also do every time I receive primers.

Some times the software that the companies use gives a different Tm than what you calculated yourself. Don't worry. Go ahead with gradient.

#5 balancer

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Posted 04 July 2010 - 06:12 PM

yeah, if you have enough sample then better run gradient. I also do every time I receive primers.

Some times the software that the companies use gives a different Tm than what you calculated yourself. Don't worry. Go ahead with gradient.


Thank you so much.;)




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