Team:British Columbia/Protocols/Sdm

From 2011.igem.org

(Difference between revisions)
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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.
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.
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 +
   '''RXN Reagent'''                      '''1x quantity'''
   '''RXN Reagent'''                      '''1x quantity'''
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   Goto line 2 for 15 more times
   Goto line 2 for 15 more times
   68°                        10 minutes
   68°                        10 minutes
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 +
<table border="3">
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<tr>
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<th>Reagents
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<th>1x Reaction Volume
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</tr>
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<tr>
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<td>ddH2O</td>
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<td>36uL</td>
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</tr>
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<tr>
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<td>10X Rxn Buffer</td>
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<td> 5uL</td>
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</tr>
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<tr>
 +
<td>dNTP (25mM</td>
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<td> 1uL</td>
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</tr>
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<tr>
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<td>Forward primer(125ng)</td>
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<td> 1uL</td>
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</tr>
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<tr>
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<td>Reverse primer(125ng)</td>
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<td> 1uL</td>
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</tr>
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<tr>
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<td>Pfu polymerase</td>
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<td> 2uL</td>
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</tr>
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<tr>
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<td>DNA template (50ng/uL)</td>
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<td> 1uL</td>
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</tr>
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<tr>
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<td>DMSO</td>
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<td> 2uL</td>
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</tr>
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<tr>
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<td>MgCl2</td>
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<td> 1uL</td>
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</table>
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 +
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Cycling Conditions:
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<table border="3">
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<tr>
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<th>Temperature
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<th>Time
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</tr>
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<tr>
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<td>95C</td>
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<td>30sec</td>
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</tr>
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<tr>
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<td>95C</td>
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<td>1 min</td>
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</tr>
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<tr>
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<td>55C</td>
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<td>1 min</td>
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</tr>
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<tr>
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<td>68C</td>
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<td>1 min/kb</td>
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</tr>
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<tr>
 +
<td></td>
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<td>Go to line 2 for 15 more times</td>
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</tr>
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<tr>
 +
<td>4C</td>
 +
 +
<td>hold</td>
 +
 +
</table>
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.  
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.  

Revision as of 01:15, 28 September 2011

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
Reagents 1x Reaction Volume
ddH2O 36uL
10X Rxn Buffer 5uL
dNTP (25mM 1uL
Forward primer(125ng) 1uL
Reverse primer(125ng) 1uL
Pfu polymerase 2uL
DNA template (50ng/uL) 1uL
DMSO 2uL
MgCl2 1uL


Cycling Conditions:

Temperature Time
95C 30sec
95C 1 min
55C 1 min
68C 1 min/kb
Go to line 2 for 15 more times
4C 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 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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