Plasmid Mini-Prep
QIAprep Spin Miniprep Kit
Isolates plasmid DNA from bacterial cells
HARVESTING
1 Centrifuge cell cultures at 2500 rpm for 10
minutes at 22ºC. This separates bacterial
cells from the growth medium to leave a
concentrated cell pellet. Carefully pour off
the supernatant (liquid above the pellet) to
leave only the pellet with the bacterial cells.
CELL LYSIS
2 Resuspend the pellet in 250 µL of Buffer P1
(resuspension buffer) and transfer solution to
new Eppendorf. Add 250 µL of Buffer P2
(lysis buffer), which lyses the cells and
denatures proteins and chromosomal DNA.
Invert samples 6 times.
NEUTRALISATION
3 Add 350 µL of Buffer N3 (neutralisation buffer). This
neutralises the alkaline conditions created by Buffer P2,
allowing the plasmid DNA to renature while precipitating the
proteins and chromosomal DNA. Invert sample 6 times.
Centrifuge samples at 13,000 rpm for 10 minutes to pellet
the cellular debris, leaving chromosomal DNA in the
supernatant.
BINDING
4 Transfer the supernatant with the plasmid
DNA to the mini-prep column. This column is
designed to bind plasmid DNA while allowing
contaminants to pass through. Centrifuge for
1 minute before discarding the supernatant.
QIAprep Spin Miniprep Kit
Isolates plasmid DNA from bacterial cells
HARVESTING
1 Centrifuge cell cultures at 2500 rpm for 10
minutes at 22ºC. This separates bacterial
cells from the growth medium to leave a
concentrated cell pellet. Carefully pour off
the supernatant (liquid above the pellet) to
leave only the pellet with the bacterial cells.
CELL LYSIS
2 Resuspend the pellet in 250 µL of Buffer P1
(resuspension buffer) and transfer solution to
new Eppendorf. Add 250 µL of Buffer P2
(lysis buffer), which lyses the cells and
denatures proteins and chromosomal DNA.
Invert samples 6 times.
NEUTRALISATION
3 Add 350 µL of Buffer N3 (neutralisation buffer). This
neutralises the alkaline conditions created by Buffer P2,
allowing the plasmid DNA to renature while precipitating the
proteins and chromosomal DNA. Invert sample 6 times.
Centrifuge samples at 13,000 rpm for 10 minutes to pellet
the cellular debris, leaving chromosomal DNA in the
supernatant.
BINDING
4 Transfer the supernatant with the plasmid
DNA to the mini-prep column. This column is
designed to bind plasmid DNA while allowing
contaminants to pass through. Centrifuge for
1 minute before discarding the supernatant.
