IGEM:UC Berkeley/2006/OverlapExtensionRxn

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Overlap extension reactions take two oligos with complementary 3' ends, and fill in the rest of the sequence with a polymerase.
Any polymerase system will work for this (Taq, Vent, Expand, Pfu...)

  1. Make 100uM stocks of the oligos (the usual concentration for stocks, it's used here as 1x)
  2. Set up per extension reaction (it's ok to make a master mix of everything except the oligos, then split it and add the oligos--just make sure to add the polymerase after the buffer and dNTPs have been added)
 34 uL ddH2O
 5 uL 2mM in each dNTPs
 5 uL 10x ThermoPol buffer
 5 uL 100 uM oligo 1
 5 uL 100 uM oligo 2
 1 uL Vent polymerase
  1. Run the "wobble" program, which is:
 step 1:  2 min at 94
 step 2:  30 sec at 55 degC
 step 3:  30 sec at 72 degC
 step 4:  Got to 2 10 times
 step 5:  hold at 4 degC forever

Note that the wobble program is basically a short PCR program without extra denaturation steps

  1. There is nothing to see on a gel, so don't bother, just go into the short DNA cleanup procedure.