General PCR

Overview

Keep everything cold all the time for better results

Protocol

  • Fill out the PCR excel spreadsheet and printout the sheet.
  • Sign-up for a PCR machine and make sure the correct PCR program exists on the machine.
  • Thaw DNA samples and controls and place on ice
  • Gather PCR cocktail ingredients (except for the taq) and PCR primers and put on ice. Make sure you have all the ingredients you need including sufficient amounts of primers.
  • Label PCR tubes or plates and place on ice in a 96 tube rack.
  • Aliquot DNA samples and controls (usually 1 or 2 µl containing between 100 to 500 ng DNA) into PCR tubes or plates. Touch pipette tip to bottom of tube when aliquoting, so DNA is at the bottom of the tube.
  • Mix the PCR cocktail using the amounts listed on the PCR excel spreadsheet in a 1.5 ml microcentrifuge tube. Always add the Taq polymerase last. Mix the complete cocktail by pipetting up and down. The Taq polymerase is normally in a glycerol solution, so it needs to be mixed well.
  • Aliquot the PCR cocktail into the PCR tubes or plates. If pipette tip touches a tube, throw it away and use a clean one.
  • Put caps on the PCR tubes or plates and seal completely.
  • Spin PCR tubes/plates.
  • Place PCR tubes/plates in PCR machine and start the PCR program.
  • Store PCR tubes/plates at 4ºC until they are analyzed on an agarose gel.