Latest revision as of 15:28, 14 September 2010

Making long term stocks | Transformation | [http://microscopy.berkeley.edu/Resources/instruction/buffers.html Buffer recipes] | Colony PCR

Colony PCR

Pick a single colony and place in 20µL of H20.
Cell lysis:
- Incubate 10 min @ 98°C
- Freeze 10 min @ -80°C
- Vortex for 2-5 min
In a PCR tube mix:
- 10µL of 2X Phusion Mastermix
- 1µL of DNA template (lysate
- XµL of Primer 1
- XµL of Primer 2
- 7µL of Nuclease free H2O
Run PCR:
- Initial Denaturation: 30s @ 98°C
- Denaturation: 10s @ 98°C
- Annealing: 30s @ Y°C
- Elongation: 30s per kb @ 72°C
- Reapeat steps 2-4, 25 to 35 times
- Final Elongation: 10 min @ 72°C
- Final Hold: ∞ @ 4°C

For Biobrick primers ([http://partsregistry.org/Part:BBa_G00100 VF2] and [http://partsregistry.org/Part:BBa_G00101 VR]) from James Brown we used the following values:
- X = 1µL
- Y = 65°C
[http://www.finnzymes.com/pdf/f531_f532_phusion_highfidelity_pcr_mastermix_datasheet_1_9_low.pdf Datasheet for the 2X Phusion Mastermix]

@@ Line 11: / Line 11: @@
 ==Method==
 *Pick a single colony and place in 20µL of H20.
+*Cell lysis:
+**Incubate 10 min @ 98°C
+**Freeze 10 min @ -80°C
+**Vortex for 2-5 min
 *In a PCR tube mix:
 **10µL of 2X Phusion Mastermix
-**1µL of DNA template
+**1µL of DNA template (lysate
 **XµL of Primer 1
 **XµL of Primer 2
 **7µL of Nuclease free H2O
 *Run PCR:
-**Initial Denaturation: 15 min @ 98°C
+**Initial Denaturation: 30s @ 98°C
 **Denaturation: 10s @ 98°C
 **Annealing: 30s @ Y°C
@@ Line 25: / Line 29: @@
 **Final Elongation: 10 min @ 72°C
 **Final Hold: ∞ @ 4°C
 ==Notes==