Team:Toulouse/Notebook/Protocols

From 2014.igem.org

(Difference between revisions)
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- Remove the supernatant
- Remove the supernatant
<br/>
<br/>
-
- Resuspend the pellet in 7.5 mL of 0.1M frozen CaCl<sub>2</sub>  
+
- Resuspend the pellet in 7.5 mL of 0.1 M frozen CaCl<sub>2</sub>  
<br/>
<br/>
-
- Centrifuge 10 minutes at 4500RPM
+
- Centrifuge 10 minutes at 4500 RPM
<br/>
<br/>
-
- Resuspend the pellet in 500µL of 0.1M CaCl<sub>2</sub>
+
- Resuspend the pellet in 500 µL of 0.1 M CaCl<sub>2</sub>
<br/>
<br/>
- Add glycerol to a final concentration of 15%
- Add glycerol to a final concentration of 15%
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- Thaw out the competent cell aliquotes for about  10 to 20 minutes
- Thaw out the competent cell aliquotes for about  10 to 20 minutes
<br/>
<br/>
-
- Add 20 to 100ng of plasmid or 3µL of kit plate DNA  
+
- Add 20 to 100 ng of plasmid or 3 µL of kit plate DNA  
<br/>
<br/>
-
<i>NB: for kit plate, resuspend the well in 10µL of sterile water</i>
+
<i>NB: for kit plate, resuspend the well in 10 µL of sterile water</i>
- Put the tubes 20 minutes in the ice
- Put the tubes 20 minutes in the ice
<br/>
<br/>
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- Put the tube 2 hours in the 37°C water bath (1 hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression
- Put the tube 2 hours in the 37°C water bath (1 hour if it concerns an ampicillin resistant strain) to allow the phenotypic expression
<br/>
<br/>
-
- Centrifuge for 1 minute at 13000RPM
+
- Centrifuge for 1 minute at 13000 RPM
<br/>
<br/>
- Remove the supernatant  
- Remove the supernatant  
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- Resuspend in 250 µL of LB medium
- Resuspend in 250 µL of LB medium
<br/>
<br/>
-
- Streak the final mix on LB agar selective medium: 200µL on one plate, 50µL on the second plate
+
- Streak the final mix on LB agar selective medium: 200 µL on one plate, 50 µL on the second plate
  </p>
  </p>
   
   
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<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)
<br/>- Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)
<br/>
<br/>
-
- Resuspend one colony per culture tube in 5mL of LB medium with antibiotic
+
- Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic
<br/>
<br/>
- Let the culture grow overnight at 37°C in a shaking incubator</p>  
- Let the culture grow overnight at 37°C in a shaking incubator</p>  
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<br>
<br>
-
<br/><I>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200µL of buffer 1 is added to resuspend the pellet, 400µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.
+
<br/><I>NB: It is possible to purify the plasmid with an alcaline lysis without any purification column. For 2 mL of culture, 200 µL of buffer 1 is added to resuspend the pellet, 400 µL of buffer 2 to allow the lysis of the cells and the denaturation of the protein and 300 µL of buffer 3 to precipitate the DNA and the proteins. The solution is then centrifuged 10 minutes at 13 000 RPM.
-
60µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse.  
+
600 µL of isopropanol is added to the supernatant and the solution is centrifuged again. The pellet is then resuspended in 100 µL of pH 7.4 TE buffer. A part of the contamination by the RNA can avoid by the addition of pH 7.4 TE buffer + 0.2 µL of RNAse.  
<br>
<br>
-
<br> <b>Buffer 1:</b> Tris 10mM pH 8 + EDTA 1mM
+
<br> <b>Buffer 1:</b> Tris 10 mM pH 8 + EDTA 1mM
-
<br> <b>Buffer 2:</b> NaOH 2mM + SDS 1%
+
<br> <b>Buffer 2:</b> NaOH 2 mM + SDS 1%
-
<br> <b>Bufer 3:</b> A COOK 3M + A COOH 15%  
+
<br> <b>Buffer 3:</b> Potassium acetate 3 M + 15% glacial acetic acid
</I></p>
</I></p>
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<p class="texte"><b>1) Digestion mix</b>
<p class="texte"><b>1) Digestion mix</b>
<br> For the vector :
<br> For the vector :
-
<br>- 5µL of miniprep plasmid  
+
<br>- 5 µL of miniprep plasmid  
<br>
<br>
-
- 2µL of each restriction enzymes
+
- 2 µL of each restriction enzymes
<br>
<br>
-
- 2µL of Green Buffer
+
- 2 µL of Green Buffer
<br>
<br>
-
- 9µL of Milli-Q water  
+
- 9 µL of Milli-Q water  
<br>
<br>
<br> For the insert :
<br> For the insert :
-
<br>- 10µL of miniprep plasmid  
+
<br>- 10 µL of miniprep plasmid  
<br>
<br>
-
- 2µL of each restriction enzymes
+
- 2 µL of each restriction enzymes
<br>
<br>
-
- 2µL of Green Buffer
+
- 2 µL of Green Buffer
<br>
<br>
-
- 4µL of Milli-Q water  
+
- 4 µL of Milli-Q water  
<br>
<br>
- Incubate 15 minutes at 37°C  
- Incubate 15 minutes at 37°C  
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- Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer
- Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer
<br>
<br>
-
- Put 20µL of sample + 6µL of marker (1kb for 1% gel and 100pb for 2%) into the well
+
- Put 20 µL of sample + 6 µL of marker (1 kb for 1% gel and 100 pb for 2%) into the well
<br>
<br>
-
- Migration for 30 min at 100V or 1hour at 50V
+
- Migration for 30 min at 100 V or 1 hour at 50V
<br>
<br>
- The revelation is made in BET (10 minutes). Then wash in water for 5 minutes
- The revelation is made in BET (10 minutes). Then wash in water for 5 minutes
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<br><br>3) Inactivation of the enzymes for the vector
<br><br>3) Inactivation of the enzymes for the vector
<br>There are two ways to inactivate the enzymes:
<br>There are two ways to inactivate the enzymes:
-
<br>- Use of DNA Clean up kit for the DNA fragment above 200pb
+
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb
<br>- Heat inactivation at 95°C for 10 minutes.</p>
<br>- Heat inactivation at 95°C for 10 minutes.</p>
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<p class="texte"><b>1) Digestion mix</b>  
<p class="texte"><b>1) Digestion mix</b>  
<br>For each part, add:  
<br>For each part, add:  
-
<br>- 5µL of miniprep plasmid  
+
<br>- 5 µL of miniprep plasmid  
-
<br>- 1µL of each restriction enzymes
+
<br>- 1 µL of each restriction enzymes
-
<br>- 2µL of Green Buffer
+
<br>- 2 µL of Green Buffer
-
<br>- 9µL of Milli-Q water  
+
<br>- 9 µL of Milli-Q water  
<br>- Incubate 15 minutes at 37°C  
<br>- Incubate 15 minutes at 37°C  
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<b>2) Inactivation of the enzymes for the vector</b>
<b>2) Inactivation of the enzymes for the vector</b>
<br>There are two ways to inactivate the enzymes:
<br>There are two ways to inactivate the enzymes:
-
<br>- Use of DNA Clean up kit for the DNA fragment above 200pb
+
<br>- Use of DNA Clean up kit for the DNA fragment above 200 pb
<br>- Heat inactivation at 95°C for 10 minutes.</p>
<br>- Heat inactivation at 95°C for 10 minutes.</p>
<p class="title2">Second step</p>
<p class="title2">Second step</p>
<p class="title3">Ligation</p>
<p class="title3">Ligation</p>
-
<p class="texte">- Mix 10µL of insert + 4µL of vector + 2µL of 10x T4 buffer + 0.5µL of T4 ligase + 3.5µL of Milli-Q water
+
<p class="texte">- Mix 10 µL of insert + 4 µL of vector + 2 µL of 10x T4 buffer + 0.5 µL of T4 ligase + 3.5 µL of Milli-Q water
<br/>
<br/>
A control without insert must be made
A control without insert must be made
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<p class="title3">Transformation</p>
<p class="title3">Transformation</p>
<p class="texte">
<p class="texte">
-
- Take 5µL of the ligation mix for 50µL of competent cells and use the <a href="http://2014.igem.org/Team:Toulouse/Notebook/Protocols#select2">Toulouse iGEM Team 2014 transformation protocol</a>.
+
- Take 5µL of the ligation mix for 50 µL of competent cells and use the <a href="http://2014.igem.org/Team:Toulouse/Notebook/Protocols#select2">Toulouse iGEM Team 2014 transformation protocol</a>.
<br/>
<br/>
- Plate the solution on selective medium overnight at 37°C.</p>
- Plate the solution on selective medium overnight at 37°C.</p>
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<p class="title2">1) Colony PCR</p>
<p class="title2">1) Colony PCR</p>
<p class="texte">
<p class="texte">
-
- Add 0.5µL of plasmid + 25µL of DreamTAQ MasterMix + 2µL of each 10µM primer (VR and VF2) + H<sub>2</sub>0 qsp 25µL and take a colony.
+
- Add 0.5 µL of plasmid + 25 µL of DreamTAQ MasterMix + 2 µL of each 10 µM primer (VR and VF2) + H<sub>2</sub>0 qsp 25 µL and take a colony.
<br/>
<br/>
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.
- Look for the number of necessary cycles and the proper temperature thanks to AmplifX or Serial Cloner 2.1 softwares.
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<p class="title2">2) Analytic digestion</p>
<p class="title2">2) Analytic digestion</p>
<p class="texte">
<p class="texte">
-
- Put a colony in 5mL of LB selective medium and wait for 6 hours
+
- Put a colony in 5 mL of LB selective medium and wait for 6 hours
<br/>
<br/>
- Make a purification thanks to the Miniprep kit
- Make a purification thanks to the Miniprep kit
<br/>
<br/>
-
- Mix 2µL of plasmid + 2µL of Fast Digest Green Buffer + 1µL of each enzyme +  Milli-Q water qsp 20µL
+
- Mix 2 µL of plasmid + 2 µL of Fast Digest Green Buffer + 1µL of each enzyme +  Milli-Q water qsp 20µL
<br/>
<br/>
-
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100V.</p>
+
- Wait 15 minutes at 37°C and put the mix on a 1% or 2% gel for 30 minutes at 100 V.</p>
<p class="title2">3) Sequencing</p>
<p class="title2">3) Sequencing</p>
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<p class="title1" id="select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</p>
<p class="title1" id="select7">Test of the pSB<sub>BS</sub>4S plasmid integration in <i>Bacillus subtilis</i> genome on the threonine site</p>
<p class="texte">
<p class="texte">
-
<br>- Plate the transformed <i>Bacillus strain</i> on a selective medium (LB + spectinomycin) overnight  
+
<br>- Plate the transformed <i>Bacillus</i> strain on a selective medium (LB + spectinomycin) overnight  
<br>- The obtained clones are then plated on different media:  Medium Competence (Thr+), Medium Competence (Thr-) and LB + Spectinomycin.  
<br>- The obtained clones are then plated on different media:  Medium Competence (Thr+), Medium Competence (Thr-) and LB + Spectinomycin.  
<br>When the plasmid is integrated, the clone can grow on minimum medium with threonine and on LB + Spectinomycin but can not grow on the minimum medium without thronine.
<br>When the plasmid is integrated, the clone can grow on minimum medium with threonine and on LB + Spectinomycin but can not grow on the minimum medium without thronine.
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<center><img src="http://2014.igem.org/wiki/images/b/b1/Installation_1.gif" width="650px"></center>
<center><img src="http://2014.igem.org/wiki/images/b/b1/Installation_1.gif" width="650px"></center>
<p class="texte">
<p class="texte">
-
<br>- Put 200µl of the different chemoattractants in the wells of the ELISA plate and pipet 15µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). The volume in the tips must be marked.<br>
+
<br>- Put 200 µl of the different chemoattractants in the wells of the ELISA plate and pipet 15 µL of each with the multichannel pipette: galactose which represents our negative control, glucose which represents our positive control and N-acetylglucosamine (NAG). The volume in the tips must be marked.<br>
-
<br><i>NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on Ceratocystis platani wall.</i><br>  
+
<br><i>NB: The NAG is the most important test because it is the monosaccharide which composes the chitin on <i>Ceratocystis platani</i> wall.<br>  
-
<br>- Put the tips with chemoattractants in 300µL of the bacterial solution in exponential growth phase in the ELISA plate.
+
<br>- Put the tips with chemoattractants in 300 µL of the bacterial solution in exponential growth phase in the ELISA plate.
<br>- Let the installation settle for 1 hour at room temperature.
<br>- Let the installation settle for 1 hour at room temperature.
<br>- After an hour, put the volume of the tips on parafilm.  
<br>- After an hour, put the volume of the tips on parafilm.  
-
<br>- Each solution is diluted 1/10,000 and 100µL is spread on LA medium.
+
<br>- Each solution is diluted 1/10,000 and 100 µL is spread on LA medium.
<br>- The plates are then incubated overnight at 37°C.</p>
<br>- The plates are then incubated overnight at 37°C.</p>
<p class="title2">Binding test</p>
<p class="title2">Binding test</p>
-
<p class="texte"><I>CBB (Chitin Binding Buffer):</I>
+
<p class="texte"><i>CBB (Chitin Binding Buffer):</i>
-
<br>- 500mM NaCl
+
<br>- 500 mM NaCl
-
<br>- 20mM Tris-HCl
+
<br>- 20 mM Tris-HCl
-
<br>- 1mM EDTA
+
<br>- 1 mM EDTA
<br>- 0,05% Triton X-100, 25°C, pH=8
<br>- 0,05% Triton X-100, 25°C, pH=8
</p>
</p>
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<p class="texte">
<p class="texte">
CAUTION : all the lab equipment must be desinfected before and after the manipulations with the fungi. <br>
CAUTION : all the lab equipment must be desinfected before and after the manipulations with the fungi. <br>
-
<br>Three different fungus strains were used : <I>Aspergillus brasiliensis, Aspergillus nidulans and Trichoderma reesei</I>
+
<br>Three different fungus strains were used : <i>Aspergillus brasiliensis</i>, <i>Aspergillus nidulans</i> and <i>Trichoderma reesei</i>
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.
<br>- The conidia can be taken by adding one drop of Tween 80 on the fungus plate.
<br>- Then the drop is mixed with 1mL of sterile water in an Eppedorf.
<br>- Then the drop is mixed with 1mL of sterile water in an Eppedorf.

Revision as of 21:58, 17 October 2014