Team:Oxford/protocols/MiniPrep: Plasmid Extraction

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<li>After growing cell cutlers 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>
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<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.

  1. 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.

  2. 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.

  3. Add 350 μl of Buffer N3 and mix the suspension immediately by inverting 6 times.

  4. Centrifuge for 10 minutes at 13000 rpm (or 17900 x g). Apply the supernatants to a spin colums

  5. Centrifuge again for 30-60 seconds and discard the flow-through.

  6. Wash the spin column by adding 500 μl of the PB buffer and centrifuge for 30-60s.

  7. Discard the flow-through and wash the spin column again by adding 750 μl of PE buffer. Centrifuge for 1 minute.

  8. Discard the flow-through and centrifuge again for 1 minute. Now place the column in a 1.5ml microcentrifuge tube.

  9. Add 50 μl of the EB buffer to the centre of the spin column. Let stand for 2 minutes and centrifuge for 1 minute.