User:Jarle Pahr/PCR

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Revision as of 08:49, 25 February 2013

http://nucleicacids.bitesizebio.com/articles/pcr-rescue/

^^ Tips on getting one band, by using one band of a first PCR as template for a second PCR:

[Phusion polymerase]

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/


http://bitesizebio.com/articles/mind-your-p%E2%80%99s-and-q%E2%80%99s-a-short-primer-on-proofreading-polymerases/


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

Polymerases

Polymerase Speed (min/kb) Fidelity Supplier Reference
Taq 1 Example ex
Phusion 0.25-0.50 Example NEB
Pfu 2 Example ex
Q5 ? Example NEB

Polymerases: http://oregonstate.edu/instruction/bb492/lectures/DNAI.html

dNTPs

Average molecular weight: 487 g/mol


Oligomer annealing

http://www.bio.net/bionet/mm/methods/1997-March/056072.html

http://www.protocol-online.org/biology-forums/posts/23721.html

http://www.oligos.com/annOligonucleotides.htm

http://www.addgene.org/plasmid_protocols/annealed_oligo_cloning/

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