Primer Annealing Temperature

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magicsiew
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Primer Annealing Temperature

Post by magicsiew » Mon Jun 07, 2010 6:23 pm

My set of primers with the forward primer with 53.0 degree of Tm and reverse primer with 64.8 degree of Tm. It is said that normally annealing temperature should be 5 to 10 degree below the primers Tm. However, in this case, even after I minus 10 degree of temperature from the reverse primer, it is still higher than the Tm of forward primer. Any suggestion on this problem? The primers were found from a literature, but inside there dint state the annealing temperature.

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JackBean
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Post by JackBean » Mon Jun 07, 2010 7:42 pm

I think it could be about 3°C lower then Tm of primer with lower Tm ;)

However, I would guess, you have some flanking regions, right?
http://www.biolib.cz/en/main/

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magicsiew
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Post by magicsiew » Mon Jun 07, 2010 11:08 pm

Means I can try with 50°C? How do check if I have some flanking region or not?

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JackBean
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Post by JackBean » Tue Jun 08, 2010 2:55 pm

yeah, I would try between 50-53°C

just anneal the primers to your sequence and you will see, whether the are 100% homologous or not. I had the same case just recently.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

magicsiew
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Post by magicsiew » Wed Jun 09, 2010 4:11 am

I tried with 55°C and it works.

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hashemyemen
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Post by hashemyemen » Sun Jun 13, 2010 3:06 am

Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.



http://en.wikipedia.org/wiki/Polymerase_chain_reaction


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JackBean
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Post by JackBean » Sun Jun 13, 2010 9:21 am

yeah, I think that's what we said
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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