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Nested PCR Internal Primer Distance

Nested PCR Internal Primer Distance - PCR - Polymerase Chain Reaction Forum

Nested PCR Internal Primer Distance - 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 02-06-2009, 10:19 PM
Pipette Filler
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Default Nested PCR Internal Primer Distance



1. When designing internal primers for nested pcr is there a rule of thumb for how internal they should be relative to the 1st round product. Currently I have chosen each primer to be about 150 bp internal to the 5' of the 1st round product with much success. However I would be interested to know if this could be reduced to less than 50.

2. Does anyone recommend having their 2nd round primer set for nested pcr having a significantly different Tm than the 1st round primer set?

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