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| | <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols/PCR purification">PCR purification</a> <br /> | | <a href="https://2015.igem.org/Team:Paris_Bettencourt/Protocols/PCR purification">PCR purification</a> <br /> |
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| − | <table style="width=35%">
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| − | <tr>
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| − | <td><b>Electroporation Protocol</b><br>
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| − | <ul>
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| − | <li>Thaw electrocompetent cells on ice
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| − | <li>Add 2µL of ligation product or 0.5µL of native plasmid to the cells
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| − | <li>Transfer the cells in an 0.2mm electroporation cuvette
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| − | <li>put the cuvette in the electroporation device and pulse the cells at 2.5kV, 200 Ohms and 25µF
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| − | <li>add 200µL of LB right after pulsing
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| − | <li>recover 2 hours at 37°C
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| − | <li>plate 200µL and 50µL of the cells on LB + erythromycin (200µg/mL), incubate overnight at 37°C
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| − | </ul>
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| − | </td>
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| − | </tr>
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| − | </table>
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| − | <table style="width=30%">
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| − | <tr>
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| − | <td><b>Analytical digestion protocol</b><br>
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| − | <ul>
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| − | <li>Prepare the following mix:
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| − | <ul>
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| − | <li>2µL 10X Digestion buffer
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| − | <li>0.5µL Eco31I
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| − | <li>0.5µL BbsI
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| − | <li>2µL of DNA (200ng)
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| − | <li>15µL water
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| − | </ul>
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| − | <li>incube 1h at 37°C
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| − | </ul>
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| − | </td>
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| − | </tr>
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| − | </table>
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| − | <table style="width=35%">
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| − | <tr>
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| − | <td><b>Electrocompetent Cells Preparation Protocol</b>
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| − | <ul>
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| − | <li>Inoculate two 250mL LB flasks with 100µL of an overnight culture of DH5α
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| − | <li>incubate until the the DO600 reach 0.5 to 0.7
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| − | <li>place the cultures on ice for 15 minutes
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| − | <li>pour the culture in cold sterile 50mL falcon tubes
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| − | <li>centrifuge them for 10 minutes at 6000rpm
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| − | <li>throw the supernatant
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| − | <li>resuspend the cells in 50mL cold distilled water
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| − | <li>centrifuge them for 10 minutes at 6000rpm
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| − | <li>throw the supernatant
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| − | <li>resuspend the cells in 25mL cold distilled water
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| − | <li>centrifuge them for 10 minutes at 6000rpm
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| − | <li>throw the supernatant
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| − | <li>resuspend the cells in 12.5mL cold 10% glycerol
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| − | <li>centrifuge them for 10 minutes at 6000rpm
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| − | <li>throw the supernatant
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| − | <li>resuspend the cells in 5mL cold 10% glycerol
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| − | <li>make aliquots of the desire volume in microcentrifuge tubes and freeze them at -80°C
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| − | </ul>
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| − | </td>
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| − | </tr>
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| − | </table>
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| − | <table style="width=%">
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| − | <tr>
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| − | <td><b>Heat Shock transformation protocol</b><br/>
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| − | <ul>
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| − | <li>Thaw frozen chemically competent cells (20µL aliquots) on ice for 10min.
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| − | <li>add 2µL of ligation product (or 0.5µL of miniprep) and incubate the cells 30sec at 42°C.
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| − | <li>put the cells back on ice for 2min.
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| − | <li>add 200µL of LB to the cells and incubate 2 hours at 37°C.
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| − | <li>plate the cells on LB + erythromycin (150µg/mL) or LB + erythromycin (10µg/mL), incubation at 37°C overnight.
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| − | </ul>
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| − | </td>
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| − | </tr>
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| − | </table>
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| − | <table style="30%">
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| − | <tr>
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| − | <td><b>Ligation Protocol</b><br>
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| − | <ul>
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| − | <li>Mix the following
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| − | <ul>
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| − | <li>vector 100ng
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| − | <li>insert 300ng
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| − | <li>T4 DNA ligase buffer 10X
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| − | <li>T4 DNA ligase
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| − | <li>up to 20µL of water
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| − | </ul>
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| − | <li>incubate 30 to 40 minutes at room temperature
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| − | </ul>
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| − | </td>
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| − | </tr>
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| − | </table>
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| − | <table style="width:35%">
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| − | <tr>
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| − | <td><b>Digestion Protocol</b><br/>
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| − | <ul>
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| − | <li>Prepare the following mix:<br/>
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| − | <ul>
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| − | <li>4μL of Enzyme 1
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| − | <li>4μL of Enzyme 2
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| − | <li>4μL of FastAP
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| − | <li>12μL of Fast Digest buffer 10X
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| − | <li>1 to 3 μg of DNA
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| − | <li>up to 120μL of water
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| − | </ul>
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| − | <li>mix by pipetting up and down
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| − | <li>incube 10min at 37°C
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| − | </ul>
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| − | </td>
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| − | </tr>
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| − | </table>
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| − |
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| − | <table style="width:75%">
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| − | <tr>
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| − | <td><b>Miniprep protocol using a QIAGEN kit </b>
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| − | <ul>
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| − | <li>centrifuge an overnight culture of cells 10min at 4krpm
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| − | <li>throw the filtrate
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| − | <li>resuspend the pellet in 250μL of Cell Resuspension Solution then mix it
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| − | <li>transfer it in a 1.5mL microcentrifuge tube
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| − | <li>add 250mL of Cell lysis solution and mix by inverting several times
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| − | <li>incube until the liquid is clear, maximum 5min
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| − | <li>add 10μL of Alkalyne protease solution, mix by inverting several times and incube 3 to 4 min
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| − | <li>add 350μL of Neutralisation Solution and mix by inverting several times
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| − | <li>centrifuge 10min at 14krpm
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| − | <li>add the supernatant in a column
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| − | <li>centrifuge 1min, 14krpm, then throw the filtrat
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| − | <li>add 750μL Washing Solution
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| − | <li>centrifuge 1min, 14krpm, then throw the filtrat
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| − | <li>add 250μL Washing Solution
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| − | <li>centrifuge 2min, 14krpm, then throw the filtrat
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| − | <li>centrifuge 1min, 14krpm
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| − | <li>transfer the column in a sterile microcentrifuge 1.5ml tube
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| − | <li>add 50μL of DNAse/RNAse free water right on the membrane of the filter, wait 1min
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| − | <li>centrifuge 1min, 14krpm
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| − | <li>throw the column, plasmid is saved in water
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| − | </ul>
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| − | </td>
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| − | </tr>
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| − | </table>
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| − |
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| − | <table>
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| − | <tr>
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| − | <td><b>Annealing Protocol</b>
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| − | <ul>
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| − | <li>Phosphorylation of the oligos
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| − | <ul>
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| − | <li> 5.6μL DNAse/RNAse free water
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| − | <li> 6.0μL o15.011 (10µM)
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| − | <li> 6.0μL o15.012 (10µM)
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| − | <li> 2.0μL 10X T4 DNA ligase buffer
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| − | <li> 0.4μL T4 PolyNucleotide Kinase
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| − | Total: 20μL
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| − | </ul>
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| − | <br/>
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| − | <li>incube 30min at 37°C
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| − | <li>add 1μL of 1M NaCl
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| − | <li>incube 5min at 95°C
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| − | <li>let the mix cool down
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| − | <li>use 2μL of the mix as a 10X solution
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| − | </ul>
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| − | </tr>
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| − | </td>
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| − | </table>
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| − |
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| − |
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| − | <table style="width:50%">
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| − | <tr>
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| − | <td>
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| − | <ul>
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| − | <b>PCR purification protocol</b>
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| − | <li>Add 5 volumes of resuspension buffer to 1 volume of PCR product in an 1.5mL microcentrifuge tube, mix by pipetting up and down
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| − | <li>Transfer in a centrifugation column
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| − | <li>Centrifuge 1 min at 14000 rpm
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| − | <li>Throw the filtration product
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| − | <li>Add 700μL of washing solution
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| − | <li>Centrifuge 1 min at 14000 rpm
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| − | <li>Throw the filtration product
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| − | <li>Add 500μL of washing solution
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| − | <li>Centrifuge 1 min at 14000 rpm
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| − | <li>Throw the filtration product
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| − | <li>Centrifuge 1 min at 14000 rpm
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| − | <li>Throw the filtration product
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| − | <li>Put the column in a sterile 1.5mL microcentrifuge tube
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| − | <li>Add 45μL of DNAse/RNAse free water on the membrane
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| − | <li>Wait 2 minutes
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| − | <li>Centrifuge 2min at 10000rpm
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| − | <li>Discard the column, DNA is saved in water
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| − | </td>
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| − | </tr>
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| − | </table>
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