QIAprep Plasmid DNA Purification

↩ Back to other protocols.

1. 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.
   
   
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.