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.

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+'''Site Directed Mutagenesis'''
 ) 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.