Team:ITESM-CEM/Project/Materials
From 2014.igem.org
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<a name="Four"><h2>Recombinant Protein Expression Protocol</h2></a> | <a name="Four"><h2>Recombinant Protein Expression Protocol</h2></a> | ||
<h4>Materials</h4> | <h4>Materials</h4> | ||
- | + | ||
- | + | <h4>Reagents</h4><br> | |
• 20 mM KH2PO4 and 10 mM KCl (pH 8) solution<br> | • 20 mM KH2PO4 and 10 mM KCl (pH 8) solution<br> | ||
• 300 mM KCl (pH 8), 20 mM Imidazole (pH 8) and 20 mM KH2PO4 solution<br> | • 300 mM KCl (pH 8), 20 mM Imidazole (pH 8) and 20 mM KH2PO4 solution<br> | ||
• Biomass recovered from the 40 mL medium<br> | • Biomass recovered from the 40 mL medium<br> | ||
• Fresh Laemmli Buffer<br> | • Fresh Laemmli Buffer<br> | ||
- | • Lysozyme (20 mg/mL)<br>< | + | • Lysozyme (20 mg/mL)<br> |
- | Equipment< | + | <h4>Equipment</h4><br> |
• Centrifuge<br> | • Centrifuge<br> | ||
• Micropipettes<br> | • Micropipettes<br> | ||
• Conical tubes<br> | • Conical tubes<br> | ||
• Centrifugal tubes<br><br> | • Centrifugal tubes<br><br> | ||
- | <h4>Procedure</h4 | + | <h4>Procedure</h4> <br> |
1. Centrifuge 40 mL medium 5000 rpm, 30 min. Remove supernatant.<br> | 1. Centrifuge 40 mL medium 5000 rpm, 30 min. Remove supernatant.<br> | ||
2. Resuspend pellet in 4 mL of a cold 300 mM KCl (pH 8), 20 mM Imidazole (pH 8) and 20 mM KH2PO4 solution.<br> | 2. Resuspend pellet in 4 mL of a cold 300 mM KCl (pH 8), 20 mM Imidazole (pH 8) and 20 mM KH2PO4 solution.<br> | ||
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6. Take 20 µL of the supernatant and mix with 20 µL of Laemmli buffer for SDS PAGE analysis.<br> | 6. Take 20 µL of the supernatant and mix with 20 µL of Laemmli buffer for SDS PAGE analysis.<br> | ||
7. The insoluble phase obtained from the previous centrifugation has to be washed twice with a 1% SDS solution and 20 mM KH2PO4 and 10 mM KCl (pH 8) solution. Then, mix insoluble phase with 20ul Laemmli buffer for SDS PAGE analysis.<br> | 7. The insoluble phase obtained from the previous centrifugation has to be washed twice with a 1% SDS solution and 20 mM KH2PO4 and 10 mM KCl (pH 8) solution. Then, mix insoluble phase with 20ul Laemmli buffer for SDS PAGE analysis.<br> | ||
- | 8. Run a SDS PAGE with iGEM ITESM CEM’s protocol.<br | + | 8. Run a SDS PAGE with iGEM ITESM CEM’s protocol.<br> |
<p style="text-align: justify; text-justify: inter-word;"> | <p style="text-align: justify; text-justify: inter-word;"> |
Revision as of 18:05, 17 October 2014
ITESM-CEM | Enzy7-K me |
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