Knight:Annealing and primer extension with Taq polymerase

This protocol uses annealing and primer extension to generate a short fragment of DNA (~100 bp) using Taq polymerase. The DNA fragment can be immediately used in a TA cloning reaction. (To proceed to a restriction digest step, purification is necessary.)

Materials

 * Two oligos which overlap by ~20 bp.

Oligo 1:   5' --- 3' Oligo 2:                                          3' --- 5'


 * PCR supermix

Procedure

 * 1) Dilute the two oligos to a concentration of 25 &mu;M using H2O.
 * 2) *General information on primer design.
 * 3) *Notes on ordering long primers.
 * 4) *Notes on efficient addition of 3'A to PCR products.
 * 5) Mix the following in a 0.6 mL sterile tube
 * 6) *9 &mu;L PCR supermix
 * 7) *0.5 &mu;L oligo 1
 * 8) *0.5 &mu;L oligo 2
 * 9) Anneal and extend the two oligos together by placing the mixture in a thermal cycler (MJ Research, PTC-200) and running the following protocol.
 * 10) 94&deg;C for 5 mins
 * 11) 94&deg;C for 30 seconds
 * 12) 55&deg;C for 30 seconds (or whatever an appropriate annealing temperature is)
 * 13) 72&deg;C for 30 seconds
 * 14) Repeat steps 2-4 2-3 cycles
 * 15) 72&deg;C for 5 mins
 * 16) Use fresh 1&mu;L PCR product in a TOPO TA cloning reaction.