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Revision as of 14:08, 21 February 2013
^^ Tips on getting one band, by using one band of a first PCR as template for a second PCR:
Finnzyme Tm calculator for use with Phusion: http://www.thermoscientificbio.com/webtools/tmc/
BiteSize Bio - The best polymerases of 2008: http://bitesizebio.com/articles/the-best-polymerases-of-2008/
Note on Phusion polymerase:
Quotes from the protocol:
The final concentration of each primer in a reaction using Phusion DNA Polymerase may be 0.2–1 μM, while 0.5 μM is recommended.
During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5–10 second denaturation at 98°C is recommended for most templates.
Annealing: Annealing temperatures required for use with Phusion tend to be higher than with other PCR polymerases. The NEB Tm calculator should be used to determine the annealing temperature when using Phusion. Typically, primers greater than 20 nucleotides in length anneal for 10–30 seconds at 3°C above the Tm of the lower Tm primer. If the primer length is less than 20 nucleotides, an annealing temperature equivalent to the Tm of the lower primer should be used. A temperature gradient can also be used to optimize
Average molecular weight: 487 g/mol