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Team:British Columbia/Protocols/Sdm
From 2011.igem.org
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.
Site Directed Mutagenesis
Procedures:
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. For greater than 10 reactions, make 1 extra sample for every 10 extra reactions (e.g. prepare n+2 master mix reactions for 20 samples, n+3 for 30, etc.)
| Reagents | 1x Reaction Volume |
|---|---|
| ddH2O | 36 μL |
| 10X Rxn Buffer | 5 μL |
| dNTP (25 mM) | 1 μL |
| Forward primer(125 ng) | 1 μL |
| Reverse primer(125 ng) | 1 μL |
| Pfu polymerase (0.5x) | 2 μL |
| DNA template (50 ng/uL) | 1 μL |
| DMSO | 2 μL |
| MgCl2 | 1 μL |
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 | Cycles |
| 95oC | 30 sec | 1 |
| 95oC | 1 min | 15 |
| 55oC | 1 min | |
| 68oC | 1 min/kb | |
| 4oC | Hold | |
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 μL Pfu was initially used, but 2 μL 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 μL of 10 μM/μL primers were initially used, but the reaction did not work until we added the suggested mass.
