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Herman Lab

PCR from Worms

  • Pick one worm and place it in a 2.5ml drop of lysis buffer in the cap of a PCR tube.  Close and centrifuge briefly to move to the bottom of the tube.
  • Freeze the tubes at –700C for 15min.  The idea is to do a freeze-crack to help liberate the DNA.  Check that the solution actually freezes.
  • Overlay with a drop of mineral oil and incubate at 600C for 60 minutes, followed by 950C for 15 minutes.
  • Cool to 40C.  Pipette 22.5ml of PCR “Master Mix” onto the top of the mineral oil overlay.  Set sample in ice bucket until all samples have been prepared.
  • Microfuge briefly to  move the Master Mix through the mineral oil overlay.  Rapidly heat the samples to 940C and cycle 30 times through a program appropriate for the template DNA and the primers you are using in the reaction:

Melting step:       940C for 30 sec (standard)

Annealing step:  ~50C below melting pt. of the primers and usually held for between 30 seconds and 1 min.

Extension step:   720C standard temp. and est. time based on length of template (approx. 1 min per 1 kb)

Analyze 10ml of each sample on a 3.0% “Metaphor agarose gel, or a 6% acrylamide gel.  Be sure to run Lambda StyI or some other molecular weight marker.

Lysis Buffer- (store on the bench top w/o proteinase K)

  • 200mg/ml proteinase K
  • 10mM Tris-Cl, pH 8.2
  • 50mM KCl
  • 2.5 mM MgCl2
  • 0.45% Tween 20
  • 0.05% gelatin

Master Mix:

Prepare a Master Mix containing the following amounts of each component PER REACTION.  Make enough Master Mix for N+1 reactions.

  • 2.5 ml 10X Amplification Buffer
  • 2.5 ml 2mM dNTPs
  • 1.5 ml 25mM MgCl2
  • 0.12 ml 5 U/ml TAQ Polymerase (0.6 Units/reaction)
  • 1.0 ml Primer Mix (25pMol/ml conc. of primer combination)
  • 15.88 ml dH2O