PCR

Materials

  • PCR tubes (one per sample, two for controls, and one extra)
  • Two 2 mL microcentrifuge tubes
  • Template
  • E. coli DNA (or other positive control)
  • Primers, forward and reverse
  • 10% BSA (LF)
  • 10x Ex Taq buffer
  • dNTP mixture
  • 5x Eppendorf Enhancer
  • Milli-Q water, autoclaved
  • Ex Taq DNA polymerase
  • Assorted pipettors and tips

Procedure

  • Open all PCR and microcentrifuge tubes and place in racks found in the PCR hood, along with all pipettors and tips (note: all pipettors should already be in hood)
  • Close the side panels and set the UV light timer for 20 minutes
  • Turn on UV light and blower for the flow hood
  • Gather all reagents and samples, placing them in an ice bath
  • Enhancer should be placed in a warm water bath to dissolve precipitate
  • Enter relevant data into the “Master Mix” calculation spreadsheet; make a print out
  • In PCR hood, prepare master mix in one 2 mL microcentrifuge tube
  • Aliquot master mix into PCR tubes
  • Transfer PCR tubes to ice bath and move to flow hood
  • Add template to each reaction tube, E. coli (or equivalent) to positive control, and PCR water to negative control
  • Place caped and labeled tubes in PCR thermocycler, then close lid
  • Turn on thermocycler, select desired PCR cycle, and begin
  • Pause thermocycler at very end of second cycle
  • Quickly add Taq polymerase to each tube (note: Do not expel polymerase from tip; it will flow out automatically. Do not allow any air bubbles to form in PCR tube.)
  • Close thermocycler lid and press “resume”
  • Store tubes at 4oC