IGEM:British Columbia/Protocols/Colony PCR: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
(→Method) |
||
| Line 25: | Line 25: | ||
## 4°C hold | ## 4°C hold | ||
* Verify PCR products on an agarose gel. | * [[iGEM:British Columbia/Protocols/Gel Verification| Verify]] PCR products on an agarose gel. | ||
Revision as of 15:26, 7 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 (details to be worked out when we get the supplies)
- 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.
