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Why should TM be same for Primers?

Why should TM be same for Primers? - PCR - Polymerase Chain Reaction Forum

Why should TM be same for Primers? - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 05-22-2008, 04:55 PM
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Question Why should TM be same for Primers?



Hi
I read that the melting temperature should be the same for both primers? Why is this?
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Old 05-24-2008, 08:22 PM
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Default Re: Why should TM be same for Primers?

Dear galgur,

The thermodynamics of a successful PCR reaction is based on the Temperature of annealing (Tm) of your primers at the appropriate sites in your template so as to produce the desired amplicon. This determines the strength of binding of your primers to the desired site. Lowering of the Tm usually causes misamplification leading to spurious products along with your desired product. Hence, Tm also maintains stringency of the reaction. The reaction mixture is held at various temperature steps in the cycle including denaturation, annealing and extension, among which the annealing temperature should be ideally a temperature that would allow the primers to bind to the desired regions, and only a single temperature can be maintained. Hence that single temperature of annealing would maintain stringency of binding for both primers only if they both share the same or around the same Tm.

There, hope this was useful!

Cheers!
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Old 11-16-2009, 05:53 AM
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Default Re: Why should TM be same for Primers?

Hi,
Is that all the primers should have some range of Tm?My forward primer Tm is 71.6 C(GC% 46.2),reverse is 70.5 C (GC%43.6).Im using 2x Pcr master mix(Fermentas)
Master mix-10 microlitre
R-primer-1 microlotre
F-primer-1 microlitre
template-1 microlitre
DW-7 micro litre
my gene is not amplified with gradient 54 C- 60 C.Non specific bands were there!!
initial denaturation at 94 C-2 min,annealing for 1 min,extention 1 min,final extention 5 min for 35 cycles.
suggest me plz....

With regards,
Chitra
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