Team:Cambridge/Protocols/ColonyPCR

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(New page: {{:Team:Cambridge/Templates/header}} {{:Team:Cambridge/Protocol Menu}} =Colony PCR= ==Materials== *2x Phusion Mastermic *Primers *Nuclease free H2O *Cell culture! ==Method== *Pick a sing...)
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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==
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*For Biobrick primers ([http://partsregistry.org/Part:BBa_G00100 VF2] and [http://partsregistry.org/Part:BBa_G00101 VF] from James Brown we used the following values:  
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*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
**X = 1µL
**Y = 65°C
**Y = 65°C
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*[http://www.finnzymes.com/pdf/f531_f532_phusion_highfidelity_pcr_mastermix_datasheet_1_9_low.pdf Datasheet for the 2X Phusion Mastermix]
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Latest revision as of 15:28, 14 September 2010

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