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Team:Oxford/protocols/MiniPrep: Plasmid Extraction
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<h1>QIAprep Plasmid DNA Purification</h1> | <h1>QIAprep Plasmid DNA Purification</h1> | ||
| + | <p>↩ Back to other <html><a href="https://2014.igem.org/Team:Oxford/protocols">protocols</a></html>.</p> | ||
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| - | <li>After growing cell | + | <li>After growing cell cultures overnight in LB, resuspend pelleted cells in 250 μl Buffer P1 and transfer it into a microcentrifuge tube. Make sure that RNase A has been added to Buffer P1 before.<BR><BR> |
<li>Add 250 μl of Buffer P2 and mix gently by inverting the tube 6 times. The cell suspension should turn blue. Try to carry on with the next step after less than 5 minutes to make sure that the lysis reaction doesn't proceed for too long.<BR><BR> | <li>Add 250 μl of Buffer P2 and mix gently by inverting the tube 6 times. The cell suspension should turn blue. Try to carry on with the next step after less than 5 minutes to make sure that the lysis reaction doesn't proceed for too long.<BR><BR> | ||
<li>Add 350 μl of Buffer N3 and mix the suspension immediately by inverting 6 times.<BR><BR> | <li>Add 350 μl of Buffer N3 and mix the suspension immediately by inverting 6 times.<BR><BR> | ||
Latest revision as of 15:11, 27 July 2014
QIAprep Plasmid DNA Purification
↩ Back to other protocols.
- After growing cell cultures overnight in LB, resuspend pelleted cells in 250 μl Buffer P1 and transfer it into a microcentrifuge tube. Make sure that RNase A has been added to Buffer P1 before.
- Add 250 μl of Buffer P2 and mix gently by inverting the tube 6 times. The cell suspension should turn blue. Try to carry on with the next step after less than 5 minutes to make sure that the lysis reaction doesn't proceed for too long.
- Add 350 μl of Buffer N3 and mix the suspension immediately by inverting 6 times.
- Centrifuge for 10 minutes at 13000 rpm (or 17900 x g). Apply the supernatants to a spin colums
- Centrifuge again for 30-60 seconds and discard the flow-through.
- Wash the spin column by adding 500 μl of the PB buffer and centrifuge for 30-60s.
- Discard the flow-through and wash the spin column again by adding 750 μl of PE buffer. Centrifuge for 1 minute.
- Discard the flow-through and centrifuge again for 1 minute. Now place the column in a 1.5ml microcentrifuge tube.
- Add 50 μl of the EB buffer to the centre of the spin column. Let stand for 2 minutes and centrifuge for 1 minute.
