Template:SBB-Protocols PCR1

Wobble Reaction
The Wobble procedure is a variation of Klenow Extension that begins with two oligonucleotides that overlap by around 20 bp on their 3' ends and uses a thermostable polymerase

29 uL water 5 uL Expand 10x Buffer 2 5 uL 10x dNTPs (2 mM in each; 0.2 mM final conc) 5 uL Oligo 1 (100uM) 5 uL Oligo 2 (100uM) 0.75 uL Expand Polymerase 1
 * Order the oligos, they don't need to be purified in any special way, smallest scale is ok
 * Make 100uM stocks, this is the concentration used directly for the reaction
 * Prepare the following reaction:

2 min at 94 10 cycles of: 30 sec at 55 30 sec at 72 (or something similar)
 * Run the wobble program, whick is:
 * There is no point in running an analytical gel afterwards, there is nothing to see
 * You'll want to run short fragment cleanups to remove the polymerase prior to digestion steps