IGEM:British Columbia/Protocols/Colony PCR: Difference between revisions
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===Method=== | ===Method=== | ||
* Make master mix of primers and other PCR components | * Make master mix of primers and other PCR components | ||
* Add Taq polymerase. | * Add Taq polymerase. | ||
* Aliquot 10uL per PCR tube. | * Aliquot 10uL per PCR tube. |
Latest revision as of 15:15, 23 August 2009
Colony PCR
Materials
- BioBrick PCR primers (G1004, G1005)
- Taq polymerase
- Buffer, Mg2+, dNTPs
- Agar plate for indexing
Equipment
- PCR
- PCR tubes
- sterile toothpick/pipet tip/innoculating loop
Method
- Make master mix of primers and other PCR components
- Add Taq polymerase.
- Aliquot 10uL per PCR tube.
- Touch toothpick/pipet tip/loop to colony, then index plate, then swirl around in PCR tube.
- Run PCR:
- 94°C for 2 mins
- 94°C for 30 secs
- 56°C for 30 secs
- 72°C for 1 min per kb of expected product, rounded up to nearest kb
- Repeat steps 2-4 24 times.
- 72°C for 3 times longer then step 4 (i.e for a expected product of 1.8 kb, step 4 will be 2 min long, step 6 will be 3*2 = 6 min long
- 4°C hold
- Verify PCR products on an agarose gel.