Difference between revisions of "Team:Paris Bettencourt/Notebook/VitaminB2"
Line 450: | Line 450: | ||
<li>Prepare the following mix:<br/> | <li>Prepare the following mix:<br/> | ||
<ul> | <ul> | ||
− | <li>4μL of Enzyme 1 | + | <li>4μL of Enzyme 1</li> |
− | <li>4μL of Enzyme 2 | + | <li>4μL of Enzyme 2</li> |
− | <li>4μL of FastAP | + | <li>4μL of FastAP</li> |
− | <li>12μL of Fast Digest buffer 10X | + | <li>12μL of Fast Digest buffer 10X</li> |
− | <li>1 to 3 μg of DNA | + | <li>1 to 3 μg of DNA</li> |
− | <li>up to 120μL of water | + | <li>up to 120μL of water</li> |
</ul> | </ul> | ||
− | <li>mix by pipetting up and down | + | <li>mix by pipetting up and down</li> |
− | <li>incube | + | <li>incube at 37°C, 10 min for FD, 45 min for regular enzymes</li> |
+ | <li>incube 10min at 70°C</li> | ||
</ul> | </ul> | ||
</td> | </td> | ||
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<td><b>Ligation Protocol</b><br> | <td><b>Ligation Protocol</b><br> | ||
<ul> | <ul> | ||
− | <li>Mix the following | + | <li>Mix the following</li> |
<ul> | <ul> | ||
− | <li>vector 100ng | + | <li>vector 100ng</li> |
− | <li>insert 300ng | + | <li>insert 300ng</li> |
− | <li>T4 DNA ligase buffer 10X | + | <li>T4 DNA ligase buffer 10X</li> |
− | <li>T4 DNA ligase | + | <li>T4 DNA ligase</li> |
− | <li>up to 20µL of water | + | <li>up to 20µL of water</li> |
</ul> | </ul> | ||
− | <li>incubate 30 to 40 minutes at room temperature | + | <li>incubate 30 to 40 minutes at room temperature</li> |
</ul> | </ul> | ||
</td> | </td> | ||
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<td><b>Heat Shock transformation protocol</b><br/> | <td><b>Heat Shock transformation protocol</b><br/> | ||
<ul> | <ul> | ||
− | <li>Thaw frozen chemically competent cells (20µL aliquots) on ice for 10min. | + | <li>Thaw frozen chemically competent cells (20µL aliquots) on ice for 10min.</li> |
− | <li>add 2µL of ligation product (or 0.5µL of miniprep) and incubate the cells 30sec at 42°C. | + | <li>add 2µL of ligation product (or 0.5µL of miniprep) and incubate the cells 30sec at 42°C.</li> |
− | <li>put the cells back on ice for 2min. | + | <li>put the cells back on ice for 2min.</li> |
− | <li>add 200µL of LB to the cells and incubate 2 hours at 37°C. | + | <li>add 200µL of LB to the cells and incubate 2 hours at 37°C.</li> |
− | <li>plate the cells on LB + erythromycin (150µg/mL) or LB + erythromycin (10µg/mL), incubation at 37°C overnight. | + | <li>plate the cells on LB + erythromycin (150µg/mL) or LB + erythromycin (10µg/mL), incubation at 37°C overnight.</li> |
</ul> | </ul> | ||
</td> | </td> | ||
Line 554: | Line 555: | ||
<td><ul> | <td><ul> | ||
<li>(ery 10µg/mL):<br> | <li>(ery 10µg/mL):<br> | ||
− | no growth | + | no growth</li> |
<li>(ery 150µg/mL):<br> | <li>(ery 150µg/mL):<br> | ||
− | lawn of bacteria</ul></td> | + | lawn of bacteria</li> |
+ | </ul></td> | ||
<td> | <td> | ||
<ul> | <ul> | ||
<li>spreading of 170µL:<br> | <li>spreading of 170µL:<br> | ||
− | 41 isolated colonies -> isolation of three transformants: T1, T2 and T3 | + | 41 isolated colonies -> isolation of three transformants: T1, T2 and T3</li> |
<li>spreading of 50µL:<br> | <li>spreading of 50µL:<br> | ||
− | 3 colonies on a lawny background | + | 3 colonies on a lawny background</li> |
</ul> | </ul> | ||
</td> | </td> | ||
<td><ul> | <td><ul> | ||
<li>(ery 10µg/mL):<br> | <li>(ery 10µg/mL):<br> | ||
− | lawn of bacteria | + | lawn of bacteria</li> |
<li>(ery 150µg/mL):<br> | <li>(ery 150µg/mL):<br> | ||
− | lawny background</ | + | lawny background</li> |
− | </td> | + | </ul></td> |
<td> no growth </td> | <td> no growth </td> | ||
</tr> | </tr> | ||
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<td><b>Electrocompetent Cells Preparation Protocol</b> | <td><b>Electrocompetent Cells Preparation Protocol</b> | ||
<ul> | <ul> | ||
− | <li>Inoculate two 250mL LB flasks with 100µL of an overnight culture of DH5α | + | <li>Inoculate two 250mL LB flasks with 100µL of an overnight culture of DH5α</li> |
− | <li>incubate until the the DO600 reach 0.5 to 0.7 | + | <li>incubate until the the DO600 reach 0.5 to 0.7</li> |
− | <li>place the cultures on ice for 15 minutes | + | <li>place the cultures on ice for 15 minutes</li> |
− | <li>pour the culture in cold sterile 50mL falcon tubes | + | <li>pour the culture in cold sterile 50mL falcon tubes</li> |
− | <li>centrifuge them for 10 minutes at 6000rpm | + | <li>centrifuge them for 10 minutes at 6000rpm</li> |
− | <li>throw the supernatant | + | <li>throw the supernatant</li> |
− | <li>resuspend the cells in 50mL cold distilled water | + | <li>resuspend the cells in 50mL cold distilled water</li> |
− | <li>centrifuge them for 10 minutes at 6000rpm | + | <li>centrifuge them for 10 minutes at 6000rpm</li> |
− | <li>throw the supernatant | + | <li>throw the supernatant</li> |
− | <li>resuspend the cells in 25mL cold distilled water | + | <li>resuspend the cells in 25mL cold distilled water</li> |
− | <li>centrifuge them for 10 minutes at 6000rpm | + | <li>centrifuge them for 10 minutes at 6000rpm</li> |
− | <li>throw the supernatant | + | <li>throw the supernatant</li> |
− | <li>resuspend the cells in 12.5mL cold 10% glycerol | + | <li>resuspend the cells in 12.5mL cold 10% glycerol</li> |
− | <li>centrifuge them for 10 minutes at 6000rpm | + | <li>centrifuge them for 10 minutes at 6000rpm</li> |
− | <li>throw the supernatant | + | <li>throw the supernatant</li> |
− | <li>resuspend the cells in 5mL cold 10% glycerol | + | <li>resuspend the cells in 5mL cold 10% glycerol</li> |
− | <li>make aliquots of the desire volume in microcentrifuge tubes and freeze them at -80°C | + | <li>make aliquots of the desire volume in microcentrifuge tubes and freeze them at -80°C</li> |
</ul> | </ul> | ||
</td> | </td> | ||
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<li>Prepare the following mix: | <li>Prepare the following mix: | ||
<ul> | <ul> | ||
− | <li>2µL 10X Digestion buffer | + | <li>2µL 10X Digestion buffer</li> |
− | <li>0.5µL Eco31I | + | <li>0.5µL Eco31I</li> |
− | <li>0.5µL BbsI | + | <li>0.5µL BbsI</li> |
− | <li>2µL of DNA (200ng) | + | <li>2µL of DNA (200ng)</li> |
− | <li>15µL water | + | <li>15µL water</li> |
− | </ul> | + | </ul></li> |
− | <li>incube 1h at 37°C | + | <li>incube 1h at 37°C</li> |
</ul> | </ul> | ||
</td> | </td> | ||
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<td><b>Electroporation Protocol</b><br> | <td><b>Electroporation Protocol</b><br> | ||
<ul> | <ul> | ||
− | <li>Thaw electrocompetent cells on ice | + | <li>Thaw electrocompetent cells on ice</li> |
− | <li>Add 2µL of ligation product or 0.5µL of native plasmid to the cells | + | <li>Add 2µL of ligation product or 0.5µL of native plasmid to the cells</li> |
− | <li>Transfer the cells in an 0.2mm electroporation cuvette | + | <li>Transfer the cells in an 0.2mm electroporation cuvette</li> |
− | <li>put the cuvette in the electroporation device and pulse the cells at 2.5kV, 200 Ohms and 25µF | + | <li>put the cuvette in the electroporation device and pulse the cells at 2.5kV, 200 Ohms and 25µF</li> |
− | <li>add 200µL of LB right after pulsing | + | <li>add 200µL of LB right after pulsing</li> |
− | <li>recover 2 hours at 37°C | + | <li>recover 2 hours at 37°C</li> |
− | <li>plate 200µL and 50µL of the cells on LB + erythromycin (200µg/mL), incubate overnight at 37°C | + | <li>plate 200µL and 50µL of the cells on LB + erythromycin (200µg/mL), incubate overnight at 37°C</li> |
</ul> | </ul> | ||
</td> | </td> | ||
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<ul> | <ul> | ||
<li>200µL:<br> | <li>200µL:<br> | ||
− | 11 single colonies | + | 11 single colonies</li> |
<li>50µL:<br> | <li>50µL:<br> | ||
− | 7 single colonies | + | 7 single colonies</li> |
</ul> | </ul> | ||
</td> | </td> | ||
Line 696: | Line 698: | ||
<ul> | <ul> | ||
<li>200µL:<br> | <li>200µL:<br> | ||
− | 32 single colonies | + | 32 single colonies</li> |
<li>50µL:<br> | <li>50µL:<br> | ||
− | 0 colony | + | 0 colony</li> |
</ul> | </ul> | ||
</td> | </td> | ||
Line 704: | Line 706: | ||
<ul> | <ul> | ||
<li>200µL:<br> | <li>200µL:<br> | ||
− | thin lawn of unrecognized bacteria with dense colonies | + | thin lawn of unrecognized bacteria with dense colonies</li> |
<li>50µL:<br> | <li>50µL:<br> | ||
− | thin lawn of unrecognized bacteria | + | thin lawn of unrecognized bacteria</li> |
</ul> | </ul> | ||
</td> | </td> | ||
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<ul> | <ul> | ||
<li>200µg/mL:<br> | <li>200µg/mL:<br> | ||
− | thin lawn of unrecognized bacteria | + | thin lawn of unrecognized bacteria</li> |
<li>150µg/mL:<br> | <li>150µg/mL:<br> | ||
− | thin lawn of unrecognized bacteria | + | thin lawn of unrecognized bacteria</li> |
</ul> | </ul> | ||
</td> | </td> | ||
Line 754: | Line 756: | ||
<ul> | <ul> | ||
<li>200µg/mL:<br> | <li>200µg/mL:<br> | ||
− | thin lawn of unrecognized bacteria | + | thin lawn of unrecognized bacteria</li> |
<li>150µg/mL:<br> | <li>150µg/mL:<br> | ||
− | thin lawn of unrecognized bacteria | + | thin lawn of unrecognized bacteria</li> |
</ul> | </ul> | ||
</td> | </td> | ||
Line 1,422: | Line 1,424: | ||
Because of a human mistake, thermocycler lid was inversed, letting the caps opening themselves when temperature increased. The PCR products were lost.<br><br> | Because of a human mistake, thermocycler lid was inversed, letting the caps opening themselves when temperature increased. The PCR products were lost.<br><br> | ||
+ | Restriction digestion of p15.01 by BbsI and BpiI(Fast Digest).<br> | ||
− | + | <img width="15%" src="https://static.igem.org/mediawiki/2015/7/77/PB_notebookB2_pic18.jpg"/><br> | |
+ | <b>Figure 11: </b><i>Electrophoresis of p15.01 after restriction digestion</i><br> | ||
+ | <i>From left to right: </i><br> | ||
+ | <i>non-digested p15.01 - p15.01 digested by BbsI (A and B) - p15.01 digested by BpiI</i> | ||
Revision as of 16:42, 22 August 2015