Fong:PCR

Preparation

 * Prepare DNA from cell sample during mid-log phase, or as extraction kit designates.
 * Use Spectrophotometer to determine concentration and total amount of extracted DNA.

Program Thermocycler

 * Use standard program.
 * Use Tannealing that is 5°C lower than the smallest Tm of the primers.
 * Use final extension time of 1 min.
 * May use extended primary melting time, to ensure full denaturation of template.
 * Use at least 30s to one min extension cycle per kb of product.

PCR Taq Mix

 * 1) Add all reagents to labeled PCR reaction tube, adding Taq polymerase last.
 * 2) Place in the thermocycler and select cycle, making any changes as noted above.
 * 3) Run PCR reaction.
 * 4) Enjoy your brand-new DNA!
 * 5) PROFT
 * 1) PROFT


 * Be sure to run at least four varying Mg2+ concentrations in order to optimize yield and fidelity.
 * Run a gel using some product and product digestion to verify fragment size and proper restriction sites.

PCR Phusion High-Fidelity Polymerase Mix

 * 1) Add all reagents to labeled PCR reaction tube, adding Phusion polymerase last.
 * 2) Place in the thermocycler and select cycle, making any changes as noted above.
 * 3) Run PCR reaction.
 * 4) Enjoy your brand-new DNA!
 * 5) PROFT
 * 1) PROFT


 * Be sure to run varying Mg2+ concentrations in order to optimize yield and fidelity.
 * Run a gel using some product and template to verify fragment size and proper restriction sites.

Contact
Yu Deng