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Team:British Columbia/Protocols/Sdm
From 2011.igem.org
iGEM Tips for Success
Things to think about when designing your project and experiments, as well as general safety rules.
Site Directed Mutagenesis
A molecular biology technique in which a mutation is created at a defined site in a DNA molecule.
Bacterial Standard Operating Protocols
How to prepare competent cells, transform your construct into competent cells, and express your protein from a lac promoter.
Yeast Standard Operating Protocols
How to transform your construct into yeast, obtain crude extract for SDS-PAGE, and perform GFP fixation for microscopy and fluorescence-activated cell sorting.
Gas Chromatography-Mass Spectrometry (GC-MS)
A method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample.
Beetle Transfer Experiments
Preliminary experiments to probe the efficiency of transferring yeast via beetle vector.
Yeast-Fungi Co-culture Experiments
Preliminary experiments to investigate the competitive interactions between yeast and the Bluestain Fungus.
1) Prepare reaction master mix for n+1 reactions, where n is the number of samples on which you wish to use SDM. For a single reaction, use the following reactants in the quantity given.
Things to think about when designing your project and experiments, as well as general safety rules.
Site Directed Mutagenesis
A molecular biology technique in which a mutation is created at a defined site in a DNA molecule.
Bacterial Standard Operating Protocols
How to prepare competent cells, transform your construct into competent cells, and express your protein from a lac promoter.
Yeast Standard Operating Protocols
How to transform your construct into yeast, obtain crude extract for SDS-PAGE, and perform GFP fixation for microscopy and fluorescence-activated cell sorting.
Gas Chromatography-Mass Spectrometry (GC-MS)
A method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample.
Beetle Transfer Experiments
Preliminary experiments to probe the efficiency of transferring yeast via beetle vector.
Yeast-Fungi Co-culture Experiments
Preliminary experiments to investigate the competitive interactions between yeast and the Bluestain Fungus.
RXN Reagent 1x quantity
dH2O 36 uL 10X Thermopol buffer 5 uL dNTP (20 mM) 1 uL FW Primer 125 ng (primers from IDT diluted to 125 ng/uL, 1 uL/reaction used) RE Primer 125 ng (primers from IDT diluted to 125 ng/uL, 1 uL/reaction used) PFU 2 uL MgCl2 1 uL DMSO 1 uL
2) Aliquot 48 uL master mix into n+1 PCR tubes. Add 50 ng template (diluted to 25 ng/uL, 2 uL/reaction used) per sample tube. Add 2 uL dH2O for water control. Be sure to clearly label the PCR tubes!
3) Place PCR tubes in thermocycler, and follow the following cycling conditions.
Temperature Time 95° 30 seconds 95° 30 seconds (Tm of primers-5°) 1 minute 68° 1-2 minute/KB Goto line 2 for 15 more times 68° 10 minutes
4) Add 0.5 uL DPNI enzyme to each sample and treat at 37° for 60 minutes or overnight to get rid of the template. Run samples on gel to confirm SDM worked.
Troubleshooting notes:
- 1 uL PFU was initially used, but 2 uL gave a brighter band on the gel
- 10 mM dNTPs were originally used, but there was no product until we increased the concentration to 20 mM.
- First reactions did not use MgCl2 or DMSO, but we found that their addition gave a more consistent, brighter band on the gel.
- PFU was used due to exonuclease activity. Only 16 cycles in total were used to prevent non-specific amplification.
- 1 uL of 10 uM/uL primers were initially used, but the reaction did not work until we added the suggested mass.
