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denaturation step of melting surve analysis


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#1 chroococcidiopsis

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Posted 16 July 2009 - 06:33 PM

Hi all~ I am new in QPCR, and i have a basic question concerning SYBR melting curve.

I am wondering why is there a default 95C denaturation prior to the drop to 60C
(i.e. [pcr profile] --> 95C x 15sec --> 60C x 1 min --> gradual increase to 95C)
what is the purpose of this step and what if i dont do that? :lol:

Many thanks !!

#2 cellcounter

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Posted 19 July 2009 - 02:09 PM

Hi all~ I am new in QPCR, and i have a basic question concerning SYBR melting curve.

I am wondering why is there a default 95C denaturation prior to the drop to 60C
(i.e. [pcr profile] --> 95C x 15sec --> 60C x 1 min --> gradual increase to 95C)
what is the purpose of this step and what if i dont do that? :D

Many thanks !!

All unique PCR products have unique nucleotide composition, and depending upon that, a unique melting curve. This step is done to see that your reaction amplifies only the product of your interest (single peak), rather than some non-specific products too (more than 1 peaks).

#3 chroococcidiopsis

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Posted 21 July 2009 - 10:55 PM

Thanks cellcounter :D
But what if i dont do the 95C denaturation? that is, after PCR, i go straight to 60C and gradual increase to 95C?

many thanks !! (& sorry if this is a dumb Q..)




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