Miniprep

From FreeBio

{Note: This protocol uses the QIAprep Spin Miniprep Kit and is used to extract and purify small amounts of plasmids from bacteria. Manufacturer's protocol can be found at http://www1.qiagen.com/literature/protocols/QIAprepMiniprep.aspx .}

Typical Conditions

1. Culture bacterial cells in 2ml selective media in 10ml plastic culture tubes overnight.
   {If the plasmid to be purified is low-copy or, like the BioBrick vectors, just bad, then use 5ml culture in 15ml VWR plastic tubes, which can be centrifuged. Also, see below: Special Conditions.} --dna1112 23:28, 20 Jun 2005 (EDT)
2. Transfer cells to 1.5ml (Eppendorf) tube by decanting or if doing biobricks, transfer to a VWR tube and centrifuge
3. Spin @ 13k rpm for 10 seconds in a microfuge to pellet.
   {If using culture tubes, use big centrifuge in side room @ 4k rpm for 5 min.}
4. Aspirate the supernatant, leaving the pellet.
5. Resuspend pellet in 250ul P1 buffer by vortexing or pipetting up and down.
6. Add 250ul P2 buffer. Mix by gentally inverting 4-6 times.
   {Note: Do NOT vortex! Vortexing can shear the genomic DNA, contaminating the plasmid prep. Do NOT allow lysing to proceed for more than 5min!}
7. Add 350ul N3 buffer. Mix immediately to prevent local precipitation by inverting 4-6 times.
8. Spin @ 13k rpm for 10min.
9. Pipette the supernatants into sky-blue QIAprep spin column.
   {Note: Be careful not to disturb the white pellet, which consists of genomic DNA and other cellular debris.}
10. Spin @ 13k rpm for 1min. Discard the flow-through, but keeping the tube.
11. Add 750ul PE buffer to column.
12. Spin @ 13k rpm for 1min and discard flow-through.
13. Spin @ 13k rpm for 1min. Place QIAprep columns into clean 1.5ml (Eppendorf) tubes.
14. Add 20-50ul EB buffer to column.
    {Note: Be careful to pipette onto the center of the membrane to ensure maximal elution.}</br>
15. Let stand for 1min and spin @ 13k rpm for 1min.
16. Store at -20C.

Special Conditions

{For low-copy plasmids and cosmids, the following protocol yields better results.} --dna1112 18:13, 7 Jul 2005 (EDT)

1. Inoculate 10-25ml culture overnight.
2. Spin down cells to pellet.
3. Resuspend pellet in 500µl Buffer P1 and transfer to a 2ml microcentrifuge tube.
4. Add 500µl Buffer P2 and gently invert the tube 4-6 times to mix.
5. Add 700µl Buffer N3 and immediately invert tube to mix.
6. Spin for 10' @ 13k rpm in microfuge.
7. Apply supernatants to a QIAprep Spin Column.
8. Centrifuge for 1' @ 13k rpm. Discard flow-through.
9. Add 500µl Buffer PB and spin for 1' @ 13k. Discard flow-through.
10. Add 750µl Buffer PE and spin for 1' @ 13k. Discard flow-through.
11. Spin again for 1' @ 13k.
12. Place QIAprep clumn in a clean 1.5ml microfuge tube. Add 50µl Buffer EB that has been pre-heated to 70°C. Let stand for 1' and spin for 1'.