Team:British Columbia/Protocols/Sdm

From 2011.igem.org

(Difference between revisions)
(→Site Directed Mutagenesis)
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Line 14: Line 14:
<tr>
<tr>
<td>ddH2O</td>
<td>ddH2O</td>
-
<td>36uL</td>
+
<td>36 μL</td>
</tr>
</tr>
<tr>
<tr>
<td>10X Rxn Buffer</td>
<td>10X Rxn Buffer</td>
-
<td> 5uL</td>
+
<td> 5 μL</td>
</tr>
</tr>
<tr>
<tr>
<td>dNTP (25mM)</td>
<td>dNTP (25mM)</td>
-
<td> 1uL</td>
+
<td> 1 μL</td>
</tr>
</tr>
<tr>
<tr>
<td>Forward primer(125ng)</td>
<td>Forward primer(125ng)</td>
-
<td> 1uL</td>
+
<td> 1 μL</td>
</tr>
</tr>
<tr>
<tr>
<td>Reverse primer(125ng)</td>
<td>Reverse primer(125ng)</td>
-
<td> 1uL</td>
+
<td> 1 μL</td>
</tr>
</tr>
<tr>
<tr>
<td>Pfu polymerase (0.5x)</td>
<td>Pfu polymerase (0.5x)</td>
-
<td> 2uL</td>
+
<td> 2 μL</td>
</tr>
</tr>
<tr>
<tr>
<td>DNA template (50ng/uL)</td>
<td>DNA template (50ng/uL)</td>
-
<td> 1uL</td>
+
<td> 1 μL</td>
</tr>
</tr>
<tr>
<tr>
<td>DMSO</td>
<td>DMSO</td>
-
<td> 2uL</td>
+
<td> 2 μL</td>
</tr>
</tr>
<tr>
<tr>
<td>MgCl2</td>
<td>MgCl2</td>
-
<td> 1uL</td>
+
<td> 1 μL</td>
</table>
</table>

Revision as of 19:19, 15 October 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

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 inthe quantity given.

Reagents 1x Reaction Volume
ddH2O 36 μL
10X Rxn Buffer 5 μL
dNTP (25mM) 1 μL
Forward primer(125ng) 1 μL
Reverse primer(125ng) 1 μL
Pfu polymerase (0.5x) 2 μL
DNA template (50ng/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.

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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