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