Team:British Columbia/Protocols/Sdm

From 2011.igem.org

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==Site Directed Mutagenesis==
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'''Procedures:'''
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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.  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.)
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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> 36 μL </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> 5 μL</td>
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</tr>
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<tr>
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<td> dNTP (25 mM) </td>
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<td> 1 μL </td>
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</tr>
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<tr>
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<td> Forward primer(125 ng) </td>
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<td> 1 μL </td>
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</tr>
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<tr>
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<td> Reverse primer(125 ng) </td>
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<td> 1 μL </td>
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</tr>
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<tr>
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<td> Pfu polymerase (0.5x) </td>
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<td> 2 μL </td>
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</tr>
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<tr>
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<td> DNA template (50 ng/uL) </td>
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<td> 1 μL </td>
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</tr>
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<tr>
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<td> DMSO </td>
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<td> 2 μL </td>
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</tr>
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<tr>
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<td> MgCl2 </td>
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<td> 1 μL </td>
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</table>
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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!
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3) Place PCR tubes in thermocycler, and follow the following cycling conditions.
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{| border="3" cellpadding="3" cellspacing="2"
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|-
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|'''Temperature''' || '''Time''' ||'''Cycles'''
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|-
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| 95<sup>o</sup>C
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| 30 sec
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| 1
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|-
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| 95<sup>o</sup>C
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| 1 min
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| rowspan="3" | 15
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|-
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| 55<sup>o</sup>C
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| 1 min
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|-
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| 68<sup>o</sup>C
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| 1 min/kb
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|-
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| 4<sup>o</sup>C
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| colspan="2" align="center"| Hold
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|}
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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.
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Troubleshooting notes:
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*1 μL Pfu was initially used, but 2 μL gave a brighter band on the gel
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*10 mM dNTPs were originally used, but there was no product until we increased the concentration to 20 mM.
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*First reactions did not use MgCl2 or DMSO, but we found that their addition gave a more consistent, brighter band on the gel.
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*Pfu was used due to exonuclease activity. Only 16 cycles in total were used to prevent non-specific amplification.
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*1 μL of 10 μM/μL primers were initially used, but the reaction did not work until we added the suggested mass.
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<html></br></html>

Latest revision as of 17:47, 18 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 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.


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