Team:Cambridge/Protocols/ColonyPCR

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

Latest revision as of 15:28, 14 September 2010