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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?
|distance , internal , nested , pcr , primer|
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