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

• 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:

 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

• Run the wobble program, whick is:

 2 min at 94
 10 cycles of:
 30 sec at 55
 30 sec at 72
 (or something similar)

• 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