PCR

For a 25 µl total volume reaction using a micropipette add to a PCR tube:

Nuclease-free H₂O 10.5 µl

Taq 2X Master Mix 12.5 µl

Forward primer 0.5 µl 10 µM solution

Reverse primer 0.5 µl 10 µM solution

DNA template 1 µl (adding sample DNA last minimizes potential contamination of the other solutions)

This is then placed in a thermocycler. General PCR cycling conditions are:

An initial 95 C 30 s

Then 30 cycles of:

95 C 20 s melting

annealing temperature (this varies with the primers used, it is very important to optimize this for your primers, usually between 55 C and 65 C although some primers can be lower) 40s

70 C 1 min extension (this can be raised to 5 min for several kbp long segments, some polymerases work better at a different temperature, 68 C - 72 C)

And a final extension of 70 C for 5 minutes.

There is a lot of optimization of these conditions based on the template DNA, primers, and polymerase enzyme used. Most commonly the annealing temperature is adjusted. Occasionally the magnesium concentration in the master mix and the extension times are also adjusted. In general there is no reason to go above 30 cycles. At this point there will theoretically be one billion copies (≈ 2³⁰) of a single starting DNA template molecule if the reaction proceeds at maximum efficiency.

Notes

What about combining robotics and molecular genetics in high school biology to build a thermocycler (transfer tubes to water baths at different temperatures and monitor for the correct temperature)?

What Links Here

• Methods ‎ (← links)
• ExoSAP Reaction ‎ (← links)