Team:Evry/Protocols/10

From 2013.igem.org

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<p>
<p>
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Transform the PTKred Plasmid with the strain of interest.
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Transform the PTKred Plasmid<a href='http://www.addgene.org/41062/' target='_blank'><small><sup>1</small></sup></a> with the strain of interest.
<br>Start an over night culture with the strain transformed with PTKred at 30°C.
<br>Start an over night culture with the strain transformed with PTKred at 30°C.
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<br>Dilute the overnight culture in a ratio of 1 mL/100 mL and add IPTG (isopropyl β-D-1-thiogalactopyranoside; final concentration of 2 mM) and Spectinomycin (60 mg/mL).
<br>Dilute the overnight culture in a ratio of 1 mL/100 mL and add IPTG (isopropyl β-D-1-thiogalactopyranoside; final concentration of 2 mM) and Spectinomycin (60 mg/mL).
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<br>Grow up to OD600: 0.5 then make <a href="https://2013.igem.org/Team:Evry/Protocols/01">electrocompetent cells</a> .  
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<br>Grow up to OD600: 0.5 then make <a href="https://2013.igem.org/Team:Evry/Protocols/01" target='_blank'>electrocompetent cells</a> .  
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Prepare the PCR fragment to be integrated flanked by 50 bp genomic sequences.
Prepare the PCR fragment to be integrated flanked by 50 bp genomic sequences.
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<br>Gel-purify the product and transform with the electrocompetent PTKred strain.
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<br><a href="https://2013.igem.org/Team:Evry/Protocols/11" target='_blank'>Gel-purify</a> the product and transform with the electrocompetent PTKred strain.
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<br>Recover in LB for 2 hours with IPTG and spectinomycin.
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<br>Recover in LB for 2 hours with IPTG.
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<br>After 2 hours incubation add the selective drug which should be inserted in the genome (for example kanamycin if you have kan R cassette inserted ) and incubate overnight in liquid culture at 30°C.
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<br>After 2 hours incubation add the selective drug which should be inserted in the genom (for example kanamycin if you have kan R cassette inserted) and incubate overnight in liquid culture at 30°C.
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<br>Next day, culture should be very turbid or the recombination did not work. Plate streak 50 μL of the overnight culture on a plate containing selection drug which in this case is Kanamycin and grow overnight at 30°C.
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<br>Next day, culture should be very turbid or the recombination did not work. Plate streak 50 μL of the overnight culture on a plate containing selection drug which in this case is kanamycin and grow overnight at 30°C.
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<br>Next Day pick a colony and make Glycerol stocks  
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<br>Next day pick a colony and make glycerol stocks  
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<p>
<p>
Inoculate the strain that you have made by Homologous Recombination in LB overnight with spectinomycin at 30°C.
Inoculate the strain that you have made by Homologous Recombination in LB overnight with spectinomycin at 30°C.
 +
<br>
<i>Adding spectinomycin in this step ensures that the plasmid will not integrate in the genome in a rare occurrence.</i>
<i>Adding spectinomycin in this step ensures that the plasmid will not integrate in the genome in a rare occurrence.</i>
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<br>Dilute 1/100 and grow at <b>42°C</b> for 4 hours and then plate sreak 50 μL of the culture om LB plate at <b>42°C</b>.   
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<br>Dilute 1/100 and grow at <b>42°C</b> for 4 hours and then plate sreak 50 μL of the culture on LB plate at <b>42°C</b>.   
<br>Next day, pick colonies and grid plate on LB then spectinomycin and grow at <b>37°C</b>.
<br>Next day, pick colonies and grid plate on LB then spectinomycin and grow at <b>37°C</b>.
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<p>Transform a cured strain with PCP20 plasmid  
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<p>
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<a href="https://2013.igem.org/Team:Evry/Protocols/02" target='_blank'>Transform</a> a cured strain with PCP20 plasmid  
<br>Grow overnight in LB with Cam at 30°C overnight.
<br>Grow overnight in LB with Cam at 30°C overnight.
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<br>Dilute 1/100 and grow for 3hrs at <b>37°C</b>.
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<br>Dilute 1/100 and grow for 3 hours at <b>37°C</b>.
<br>Streak plate on LB plates and grow over night at <b>42°C</b>.   
<br>Streak plate on LB plates and grow over night at <b>42°C</b>.   
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<br>Repeart the grid plating as in the diagram  
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<br>Repeart the grid plating as in the following diagram:
<h1> AJouter image</h1>
<h1> AJouter image</h1>
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</p>
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<div id="citation_box">
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<p id="references">Reference:</p>
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<ol>
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  <li>http://www.addgene.org/41062/</li>
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</ol>
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</div>
</div>
</div>

Latest revision as of 10:38, 6 September 2013

Iron coli project

Homologous Recombination

Aim

Preparation

Be careful, all recombination protocols should be done at 30°C unless indicated otherwise !

Strain Preparation

Transform the PTKred Plasmid1 with the strain of interest.
Start an over night culture with the strain transformed with PTKred at 30°C.
Dilute the overnight culture in a ratio of 1 mL/100 mL and add IPTG (isopropyl β-D-1-thiogalactopyranoside; final concentration of 2 mM) and Spectinomycin (60 mg/mL).
Grow up to OD600: 0.5 then make electrocompetent cells .

Integration

Prepare the PCR fragment to be integrated flanked by 50 bp genomic sequences.
Gel-purify the product and transform with the electrocompetent PTKred strain.
Recover in LB for 2 hours with IPTG.
After 2 hours incubation add the selective drug which should be inserted in the genom (for example kanamycin if you have kan R cassette inserted) and incubate overnight in liquid culture at 30°C.
Next day, culture should be very turbid or the recombination did not work. Plate streak 50 μL of the overnight culture on a plate containing selection drug which in this case is kanamycin and grow overnight at 30°C.
Next day pick a colony and make glycerol stocks

Elimination of PTKred plasmid

Inoculate the strain that you have made by Homologous Recombination in LB overnight with spectinomycin at 30°C.
Adding spectinomycin in this step ensures that the plasmid will not integrate in the genome in a rare occurrence.
Dilute 1/100 and grow at 42°C for 4 hours and then plate sreak 50 μL of the culture on LB plate at 42°C.
Next day, pick colonies and grid plate on LB then spectinomycin and grow at 37°C.

Flip Recombinase and Anti-biotic Resistance Gene

Transform a cured strain with PCP20 plasmid
Grow overnight in LB with Cam at 30°C overnight.
Dilute 1/100 and grow for 3 hours at 37°C.
Streak plate on LB plates and grow over night at 42°C.
Repeart the grid plating as in the following diagram:

AJouter image

Reference:

  1. http://www.addgene.org/41062/