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Team:Imperial College London/Protocols General
From 2011.igem.org
Protocols
This page lists all the protocols used in our project. We have classified them into five main categories as follow.
General
Cell Transformation
DNA Rehydration
Colony PCR
- 20.75 ul H2O
- 2.5 ul Pfu Buffer
- 0.25 ul dNTP
- 0.5 ul forward primer
- 0.5 ul reverse primer
- 0.5 ul Pfu Cx polymerase
Make up a master mix of everything except polymerase and aliquot into PCR tubes. Pick colonies from your plate, spot onto your reference grid plate and then pipette up and down in the PCR tube. The high denaturation temperature of PCR lyses the cells so that the DNA contents can be used as template for sequencing primers to anneal to and polymerase to extend, so you can verify that the colonies contain your plasmid based on analytical gel electrophoresis.
CPEC assembly
Make up 50 ul reactions with 5 ul DNA total with each part to be assembled at equimolar concentrations (ie. make appropriate dilutions)
-33.5 ul ddH2O
-10 ul Phusion HF buffer (5x)
-1 ul dNTP mix (40 mM)
-5 ul DNA (1-50 ng)
-0.5 Phusion high fidelity DNA polymerase (2 U/ul-1)
Thermostability Assay
- Take 50ul of crude lysate and heat for 2 hours at the following temperatures (degrees centigrade) in a PCR thermocycler. 35.3, 37.1, 39.7, 44.1, 48.5, 52.5, 56.7, 57.1, 59.1, 61.3, 61.7, 65.7, 66.3, 69.7, 70.9, 72.5, 74.1, 75.3, 79.1, 83.7, 88.8, 92.3, 95.1, 96.5
- Dilute 30ul of heat treated samples in 170ul of lysis buffer and measure fluorescence on plate reader.
