Team:Northwestern/Protocols/PCR
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Latest revision as of 21:35, 28 September 2012
Polymerase Chain Reaction
Procedure
PCR Amplification (50 ul)
- To a small PCR tube (on ice) add:
- enough water to fill to 50 ul (19 uL)
- 1 ul of template DNA
- 2.5 ul each of 10 uM forward and reverse primers
- 25 ul of PCR Master Mix
- Quickly transfer PCR tubes from ice to a preheated thermocycler with the following conditions:
- initial denaturation: 94°C for 30 seconds
- 25-35 cycles: 94°C for 15-30 seconds (we did 30 sec)
- 45-68°C for 15-60 seconds (we did 60°C for 30 sec)
- 68°C for 1min/kb (we did 2 min)
- final extension: 68°C for 5 min
- hold: 4°C for as long as necessary
PCR Purification We used the QIAquick PCR Purification Kit from Qiagen.
- Add 5 volumes of Buffer PB to 1 volume of PCR sample and mix. Apply sample to a spin column and centrifuge at 13,000 rpm for 30-60 seconds.
- Discard flow-through.
- Place the column back into the same tube.
- Wash with 0.75 mL Buffer PE and centrifuge at 13,000 rpm for 30-60 seconds.
- Discard flow-through and place the column back into the same tube.
- Dry spin for an additional 1 minute at 13,000 rpm.
- Place column in a clean 1.5 mL microcentrifuge tube. Add 50 uL of nuclease free water to the center of the membrane.
- Let sit at room tempature for 10 minutes.
- Centrifuge the column at 13,000 rpm for one minute to elute.
- Store at -20°C.