Team:British Columbia/Protocols/Sdm

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Team: British Columbia - 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.


Site Directed Mutagenesis

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.

  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.