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