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thinker

Member Since 31 Jan 2012
Offline Last Active May 01 2013 12:30 PM

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Topics I've Started

Pair of primers (SYBR) with more than one amplicon product(?)

27 March 2013 - 04:11 AM

Hi everyone,

I have a question.

I am trying to design a pair of primers in a repetitive sequence, but i am facing the issue below:

The forward primer is ok and align in only one site in the sequence.

But the reverse primer align in four sites downstream the sequence amplifying 4 amplicons: 213bp, 425bp, 637bp and 849bp.

My question is: at the end of PCR, how many amplicons will I have?

Theoretically I know that I will have 4 amplicons, but I was thinking if the first aligment of the reserve primer (213bp) will allow the other bigger amplicons to be produced.

What do you think about it?

Thanks,

Thinker

PCR with more than one primer

07 July 2012 - 10:13 AM

Could someone help me?

Is it possible to perform a pcr with four pair of primers in the same tube, and then, to check in an agarose gel, the products formed?

PCR optimization: PCR vs qPCR

08 June 2012 - 05:31 AM

The correct way to start a qPCR is to find the better Tm of the primer, isn´t it?

So, we perform a gradient of conventional PCRs to discover that. But the concentrations of reagents of a conventional PCR is different from those of qPCR and the concentrations of reagents change the Tm of the primer.

My question is: how can I labor with these differences? Is this relevant? Or the same Tm of the primer in a conventional PCR will be the same in a qPCR?

What temp the lid of the thermocycler must be set?

25 May 2012 - 12:54 PM

Hello Guys,

I have to perfom a bisulfite conversion of my sample of DNA.

The instruction of the manufacturer protocol is:
"Thermal cycler with heated lid (since the bisulfite reaction is not overlaid
with mineral oil, only thermal cyclers with heated lids are suitable for this
procedure)"

and

"IMPORTANT: Since the bisulfite reaction is not overlaid with mineral oil,
only thermal cyclers with heated lids are suitable for this procedure. It is
important to use PCR tubes that close tightly."

What is the temperature that the lid should remain during cycling?

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