PCR from Worms

Ø      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.

Ø      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.

Ø      Overlay with a drop of mineral oil and incubate at 60⁰C for 60 minutes, followed by 95⁰C for 15 minutes.

Ø      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.

Ø      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)

Ø      Analyze 10ml of each sample on a 3.0% “Metaphor 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 dH₂O