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Is this primer okay?

primer PCR

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4 replies to this topic

#1 JianxiaoHuang

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Posted 01 August 2013 - 05:45 AM

hi everbody

I need to get rid of dimer of primer and ensure the Tm value is about 60oC, so I add some bases, which dose not pair with the template in the middle, to this primer.

can anybody tell me whether or not this primer works in qPCR.

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#2 bob1

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Posted 01 August 2013 - 12:46 PM

I'm no expert on qPCR, so you can take this any way you want, but for maximal efficiency you would need to use a primer that binds completely to the target, adding bases inside primers is never a good idea unless you want to generate mutants (e.g. site directed mutagenesis), as these additions lower the efficiency of the PCR.



#3 JianxiaoHuang

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Posted 01 August 2013 - 01:27 PM

I'm no expert on qPCR, so you can take this any way you want, but for maximal efficiency you would need to use a primer that binds completely to the target, adding bases inside primers is never a good idea unless you want to generate mutants (e.g. site directed mutagenesis), as these additions lower the efficiency of the PCR.

Thank you, bob1



#4 phage434

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Posted 01 August 2013 - 05:46 PM

That primer Might work, if you lower your annealing temperature. But I would definitely redesign it given an opportunity. The mismatches are at the 5' end, so are much less likely to cause problems, but you have only 15 bp or so of correctly matched bases, which is not enough to get good specificity and a decent annealing temperature.



#5 JianxiaoHuang

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Posted 01 August 2013 - 08:15 PM

That primer Might work, if you lower your annealing temperature. But I would definitely redesign it given an opportunity. The mismatches are at the 5' end, so are much less likely to cause problems, but you have only 15 bp or so of correctly matched bases, which is not enough to get good specificity and a decent annealing temperature.

As you said, I redesigned a better one.Thank you for your explanation.







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