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Team:Sumbawagen/Notebook/protocol6
From 2014.igem.org
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<p>Spin column based purification was used. The kit is Plasmid mini kit (Nippon Genetics). Standard protocols from the kit were followed by adaptation because we have no high speed microcentrifuge, but only a 6,000 rpm small microcentrifuge.</p> | <p>Spin column based purification was used. The kit is Plasmid mini kit (Nippon Genetics). Standard protocols from the kit were followed by adaptation because we have no high speed microcentrifuge, but only a 6,000 rpm small microcentrifuge.</p> | ||
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1. Add 2 ml of E. coli overnight culture into 2 ml polypropylene tube. Centrifuge for 6,000 rpm; 5 minutes. Remove the supernatant. | 1. Add 2 ml of E. coli overnight culture into 2 ml polypropylene tube. Centrifuge for 6,000 rpm; 5 minutes. Remove the supernatant. | ||
2. Add 200 ul of mP1; vortexing. Add 200 ul of mP2; invert the tube 10 times. Incubate at 2 minutes at room temperature. Add 300 ul of mP3; invert the tube. | 2. Add 200 ul of mP1; vortexing. Add 200 ul of mP2; invert the tube 10 times. Incubate at 2 minutes at room temperature. Add 300 ul of mP3; invert the tube. | ||
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10. Obtain the plasmid as the flowthrough. | 10. Obtain the plasmid as the flowthrough. | ||
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<p>2. Samples were incubated at 37 oC for more than 1 hour (typically 3 hours).</p> | <p>2. Samples were incubated at 37 oC for more than 1 hour (typically 3 hours).</p> | ||
<p>3. When necessary, samples were electrophoresed using protocol “3. Electrophoresis”(typically 3 hours).</p> | <p>3. When necessary, samples were electrophoresed using protocol “3. Electrophoresis”(typically 3 hours).</p> | ||
<p>4. Samples were purified using protocol “4. DNA purification”.</p> | <p>4. Samples were purified using protocol “4. DNA purification”.</p> | ||
Revision as of 00:55, 18 October 2014
Team:Sumbawagen/Team
From 2014.igem.org
Notebook – Protocol – 6. Plasmid purification
Spin column based purification was used. The kit is Plasmid mini kit (Nippon Genetics). Standard protocols from the kit were followed by adaptation because we have no high speed microcentrifuge, but only a 6,000 rpm small microcentrifuge.
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1. Add 2 ml of E. coli overnight culture into 2 ml polypropylene tube. Centrifuge for 6,000 rpm; 5 minutes. Remove the supernatant.
2. Add 200 ul of mP1; vortexing. Add 200 ul of mP2; invert the tube 10 times. Incubate at 2 minutes at room temperature. Add 300 ul of mP3; invert the tube.
3. Immediately centrifuge for 6,000 rpm; 5 minutes.
4. Load the supernatant onto spin column. Centrifuge 6,000 rpm; 1 minute. Discard the flowthrough.
5. Add 400 ul of mP4. Centrifuge 6,000 rpm; 1 minute. Discard the flowthrough.
6. Add 600 ul of mP5. Centrifuge 6,000 rpm; 1 minute. Discard the flowthrough.
7. Centrifuge 6,000 rpm; 5 minute to dry the column.
8. Replace collecting tube with clean, sterlized 1,5 ml size polypropylene tube.
9. Add 50 ul of mP6. Incubate at room temperature for 2 minutes. Centrifuge for 6,000 rpm; 5 minutes.
10. Obtain the plasmid as the flowthrough.
2. Samples were incubated at 37 oC for more than 1 hour (typically 3 hours).
3. When necessary, samples were electrophoresed using protocol “3. Electrophoresis”(typically 3 hours).
4. Samples were purified using protocol “4. DNA purification”.
