PCR from Worms

  1. Pick one worm and place it in a 2.5ul drop of lysis buffer in the cap of a PCR tube.  Close and centrifuge briefly to move to the bottom of the tube.
  2. Freeze the tubes at –800C for 15min.  The idea is to do a freeze-crack to help liberate the DNA.  Check that the solution actually freezes. (Step can be repeated for better results if necessary)
  3. Incubate at 600C for 60 minutes, followed by 950C for 15 minutes. Cool to 40C. Use program "Worm lysis" on thermocycler.
  4. Pipette 22.5ul of PCR “Master Mix” onto each sample.  Set sample in ice bucket until all samples have been prepared. Microfuge briefly.  
  5. Run PCR  program appropriate for the template DNA and the primers you are using in the reaction (See Standard PCR Conditions protocol).
  6. Analyze 5ul of each sample on a 2.0% agarose gel. Be sure to run Lambda StyI or some other molecular weight marker.

Lysis buffer (store at room temperature WITHOUT proteinase K): 

  • 10mM Tris-Cl, pH 8.2 (100 uL of 1 M in 10 mL) 
  • 50mM KCl (500 uL of 1 M in 10 mL)
  • 2.5 mM MgCl2 (25 uL of 1 M in 10 mL)
  • 0.45% Tween 20 (450 uL of 10% in 10 mL)
  • 0.05% gelatin (250 uL of 2% in 10 mL)
  • Water (Final volume to 10 mL)
  • 100ug/ml proteinase K (.5 uL per 100 uL Lysis Buffer)

Master Mix:

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

  • 15.88 uL dH2O
  • 1X Buffer (5 uL)
  • 2.5mM dNTPs (0.5 uL of 10 mM)
  • .6 Units/reaction polymerase (.12 uL)
  • .5 uM F. primer (0.5 ul 25 pM/uL)
  • .5 uM R. primer (0.5 ul 25 pM/uL)

*optional .5 mM MgCl2 (1.5 ul 25 mM MgCl2)