Template:SBB-Protocols PCR1

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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