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Team:INSA Toulouse/contenu/lab practice/notebook/protocols/charac recomb
From 2013.igem.org
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| - | <p class="texte"><b>In vitro recombination test Bxb1<SUP> | + | <p class="texte"><b>In vitro recombination test Bxb1<SUP>1</SUP> recombinase</b><br> |
<div class="list"> | <div class="list"> | ||
<ol > | <ol > | ||
| - | <li>Recombination reactions were assembled on ice with 100 ml<SUP> | + | <li>Recombination reactions were assembled on ice with 100 ml<SUP>2</SUP> of Bxb1 recombinase extraction solution and 20 ml of solution containing Rec buffer 1<SUP>3</SUP> and 60 ng<SUP>4</SUP> the gate plasmid.</li> |
| - | <li>Reactions were incubated at 37°C<SUP> | + | <li>Reactions were incubated at 37°C<SUP>5</SUP>.</li> |
| - | <li>Taking sample (20 µl) for different time from 15 min to 5 hours<SUP> | + | <li>Taking sample (20 µl) for different time from 15 min to 5 hours<SUP>6</SUP> and heat inactivated at 75°C for 10min.</li> |
<li>Reaction taking samples were transformed into E.coli K12-DH5.1.</li> | <li>Reaction taking samples were transformed into E.coli K12-DH5.1.</li> | ||
<li>Spread out the cells on LB agar plate containing the antibiotic to which the gate plasmid contains the resistance gene.</li> | <li>Spread out the cells on LB agar plate containing the antibiotic to which the gate plasmid contains the resistance gene.</li> | ||
| - | <li>The plate was incubated overnight at 37°C<SUP> | + | <li>The plate was incubated overnight at 37°C<SUP>5</SUP>.</li> |
<li>In order to RFP expressed can detected by human eye, the plate was exposed after in room temperature for 16 hours up to 32 hours.</li> | <li>In order to RFP expressed can detected by human eye, the plate was exposed after in room temperature for 16 hours up to 32 hours.</li> | ||
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| - | <td style="border-bottom:4px solid #e5e6e6; border-top-left-radius:9px;">Recombinase(s) tested</td> | + | <td style="border-bottom:4px solid #e5e6e6; border-top-left-radius:9px;"><SUP>1 </SUP>Recombinase(s) tested</td> |
| - | <td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;">Buffer</td> | + | <td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><SUP>2 </SUP>Buffer</td> |
| - | <td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;">Protein volume (mL)</td> | + | <td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><SUP>3 </SUP>Protein volume (mL)</td> |
| - | <td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;">Plasmid DNA (ng) | + | <td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><SUP>4</SUP>Plasmid DNA (ng) |
</td> | </td> | ||
| - | <td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;">Incubation Temperature (°C) | + | <td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><SUP>5 </SUP>Incubation Temperature (°C) |
</td> | </td> | ||
| - | <td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;">Incubation Time | + | <td style="border-bottom:4px solid #e5e6e6; border-top-right-radius:9px;"><SUP>6 </SUP>Incubation Time |
</td> | </td> | ||
</tr> | </tr> | ||
Revision as of 15:47, 4 October 2013
Notebook
Protocols
In vitro recombinase characterization protocol
Recombinase overexpression and extraction
- Pre-culture overnight of the strain containing recombinase plasmid at 37°C.
- Culture at 37°C for 4 hours.
- Centrifuge the culture for 3 min at 13000 rpm.
- The cell pellets were resuspended in 600µl Rec buffer 1 (for Bxb1 and FimE recombinases) or Rec buffer 2 (for Tp901 and PhiC31 recombinases).
- Sonication of the cells for three bursts of 30s.
- Centrifugation for 15 min at 13000 rpm, 4°C, the supernatant containing proteins (recombinases) was collected and incubated on ice.
- Rec buffer 1 : 20 mM Tris, pH 7.5, 10mM EDTA, 25mM NaCl, 10mM Spermidine, 1mM DTT and 0.1g.mol-1 BSA.
- Rec buffer 2 : 20 mM Tris, pH 7.5, 1mM EDTA, 1M NaCl.
- Notes : For overexpression of Tp901 and Bxb1 from Dual Controller plasmid of Bonnet, 20 ng/ml of aTc and 1% of arabinose were added to the culture in order to activate the pTetO and the pBAD promoters.
In vitro recombination test Bxb11 recombinase
- Recombination reactions were assembled on ice with 100 ml2 of Bxb1 recombinase extraction solution and 20 ml of solution containing Rec buffer 13 and 60 ng4 the gate plasmid.
- Reactions were incubated at 37°C5.
- Taking sample (20 µl) for different time from 15 min to 5 hours6 and heat inactivated at 75°C for 10min.
- Reaction taking samples were transformed into E.coli K12-DH5.1.
- Spread out the cells on LB agar plate containing the antibiotic to which the gate plasmid contains the resistance gene.
- The plate was incubated overnight at 37°C5.
- In order to RFP expressed can detected by human eye, the plate was exposed after in room temperature for 16 hours up to 32 hours.
| 1 Recombinase(s) tested | 2 Buffer | 3 Protein volume (mL) | 4Plasmid DNA (ng) | 5 Incubation Temperature (°C) | 6 Incubation Time |
| Bxb1 | 100 | Buffer1 | 100 | 37 | 15min to 5h |
| Tp901 | 100 | Buffer2 | 100 | 37 | 3h to 10h |
| Bxb1 & Tp901 | 100 each | Buffer2 | 220 | 37 | 3h to 10h |
| PhiC31 | 100 | Buffer2 | 100 | 30 | 3h to 10h |
| FimE | 100 | Buffer1 | 100 | 37 | 3h to 12h |
| PhiC31 & FimE | 100 each | Buffer2 | 220 | 30 | 3h to 12h |
