Team:British Columbia/Protocols/Sdm

From 2011.igem.org

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{{Template:Protocols}}
{{Template:Protocols}}
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'''Site Directed Mutagenesis'''
 
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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.
 
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  '''RXN Reagent'''                     '''1x quantity'''
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  dH2O                            36 uL
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==Site Directed Mutagenesis==
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  10X Thermopol buffer            5 uL
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  dNTP (20 mM)                     1 uL
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'''Procedures:'''
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  FW Primer                        125 ng (primers from IDT diluted to 125 ng/uL, 1 uL/reaction used)
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  RE Primer                        125 ng (primers from IDT diluted to 125 ng/uL, 1 uL/reaction used)
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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.)
-
  PFU                              2 uL
+
 
-
  MgCl2                            1 uL
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<table border="3">
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  DMSO                             1 uL
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<tr>
 +
<th> Reagents
 +
<th> 1x Reaction Volume
 +
</tr>
 +
<tr>
 +
<td> ddH2O </td>
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<td> 36 μL </td>
 +
</tr>
 +
<tr>
 +
<td> 10X Rxn Buffer </td>
 +
<td> 5 μL</td>
 +
</tr>
 +
<tr>
 +
<td> dNTP (25 mM) </td>
 +
<td> 1 μL </td>
 +
</tr>
 +
<tr>
 +
<td> Forward primer(125 ng) </td>
 +
<td> 1 μL </td>
 +
</tr>
 +
<tr>
 +
<td> Reverse primer(125 ng) </td>
 +
<td> 1 μL </td>
 +
</tr>
 +
<tr>
 +
<td> Pfu polymerase (0.5x) </td>
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<td> 2 μL </td>
 +
</tr>
 +
<tr>
 +
<td> DNA template (50 ng/uL) </td>
 +
<td> 1 μL </td>
 +
</tr>
 +
<tr>
 +
<td> DMSO </td>
 +
<td> 2 μL </td>
 +
</tr>
 +
<tr>
 +
<td> MgCl2 </td>
 +
<td> 1 μL </td>
 +
</table>
 +
 
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!
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.
3) Place PCR tubes in thermocycler, and follow the following cycling conditions.
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  '''Temperature'''                 '''Time'''
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{| border="3" cellpadding="3" cellspacing="2"
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  95°                        30 seconds
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|-
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  95°                        30 seconds
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|'''Temperature''' || '''Time''' ||'''Cycles'''
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  (Tm of primers-5°)          1 minute
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|-
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  68°                        1-2 minute/KB
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| 95<sup>o</sup>C
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  Goto line 2 for 15 more times
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| 30 sec
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  68°                        10 minutes
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| 1
 +
|-
 +
| 95<sup>o</sup>C
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| 1 min
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| rowspan="3" | 15
 +
|-
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| 55<sup>o</sup>C
 +
| 1 min
 +
|-  
 +
| 68<sup>o</sup>C
 +
| 1 min/kb
 +
|-
 +
| 4<sup>o</sup>C
 +
| colspan="2" align="center"| 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.  
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:  
Troubleshooting notes:  
-
*1 uL PFU was initially used, but 2 uL gave a brighter band on the gel
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*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.
*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.
*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.
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*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.
+
*1 μL of 10 μM/μL primers were initially used, but the reaction did not work until we added the suggested mass.
 +
<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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