Team:Evry/Protocols/10
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- | Transform the PTKred Plasmid with the strain of interest. | + | 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). | ||
- | <br>Grow up to OD600: 0.5 then make <a href="https://2013.igem.org/Team:Evry/Protocols/01">electrocompetent cells</a> . | + | <br>Grow up to OD600: 0.5 then make <a href="https://2013.igem.org/Team:Evry/Protocols/01" target='_blank'>electrocompetent cells</a> . |
</p> | </p> | ||
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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. | ||
- | <br>Gel-purify the product and transform with the electrocompetent PTKred strain. | + | <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. |
- | <br>Recover in LB for 2 hours with IPTG | + | <br>Recover in LB for 2 hours with IPTG. |
- | <br>After 2 hours incubation add the selective drug which should be inserted in the | + | <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. |
- | <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 | + | <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. |
- | <br>Next | + | <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> | ||
- | <br>Dilute 1/100 and grow at <b>42°C</b> for 4 hours and then plate sreak 50 μL of the culture | + | <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>. | ||
</p> | </p> | ||
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- | <p>Transform a cured strain with PCP20 plasmid | + | <p> |
+ | <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. | ||
- | <br>Dilute 1/100 and grow for | + | <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>. | ||
- | <br>Repeart the grid plating as in the diagram | + | <br>Repeart the grid plating as in the following diagram: |
<h1> AJouter image</h1> | <h1> AJouter image</h1> | ||
</p> | </p> | ||
+ | |||
+ | <div id="citation_box"> | ||
+ | <p id="references">Reference:</p> | ||
+ | <ol> | ||
+ | <li>http://www.addgene.org/41062/</li> | ||
+ | </ol> | ||
+ | </div> | ||
</div> | </div> |
Latest revision as of 10:38, 6 September 2013
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:
- http://www.addgene.org/41062/