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 ÒMaster MixÓ 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% ÒMetaphor agarose gel, or a 6% acrylamide gel.  Be sure to run lstyI 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 dH2