A protocol of Miniprep
Isolation of
Plasmid by QIAprep
Spin Miniprep Kit
* All protocol steps should be carried out at room
temperature.
* Check that Ethanol has been added to PE
buffer and RNase was added to Buffer P1.
* Buffer P1 must be stored in the fridge.
* Buffers that are used in this
protocol are: P1 (with RNase, in the fridge), P2
(lysis buffer contains SDS and NaOH), N3 (Neutralization contains potassium
acetate), Buffer PB and Buffer PE (with ethanol).
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(1) Take the O/N (LB/antibiotic)
culture from the shaking incubator and under aseptic condition, pipet 450µl of it + 50 µl sterile Glycerol in
an autoclaved eppendorf and keep it at -80C as a
Glycerol stock.
(2) Centrifuge the remaining O/N
culture at 3000 for 10min at 4°C to precipitate the cells into the
bottom. Decant the supernatant and keep the pellet.
(3) Resuspend pelleted bacterial cells in 250 µl Buffer P1 and
transfer to a microcentrifuge tube.
(4) Add
250 µl Buffer P2 (SDS/NaOH lysis
buffer) and invert the tube gently 4–6 times to mix. Do not vortex, as this will result in shearing of genomic
DNA. Do not allowthe lysis reaction to
proceed for more than 5 min.
(5) Add 350 µl Buffer N3 (Neutralization buffer of
potassium acetate) and invert the tube immediately but gently 4–6 times. To
avoid localized precipitation, immediately after addition of Buffer N3,
mix the solution gently but
thoroughly.
(6) Centrifuge for 10 min at 13000 rpm. Keep the supernatant and
transfer it to to
the QIAprep column.
(7) Centrifuge
for 30–60 s at
13000 rpm. Discard the flow-through.
(8) Wash the QIAprep
spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s at 13000
rpm. Discard the flow-through. Although this step isn't
important for some host transformants like DH5α,
it is important for a low copy plasmid such as pET30a we would like to isolate
it.
(9) Wash QIAprep
spin column by adding 0.75 ml Buffer PE and
centrifuging for 30–60 s at 13000 rpm. Discard the flow-through.
(10) Centrifuge for an additional 1
min to remove residual wash buffer. IMPORTANT:
Residual wash buffer will not be completely removed unless theflow-through
is discarded before this additional centrifugation. Residual ethanol from
Buffer PE may inhibit subsequent enzymatic reactions.
(11)
Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB
(10 mM Tris·Cl, pH 8.5) or
50 µl water to the center of
each QIAprep column, let stand for 1 min, and
centrifuge for 1 min.
Determine the concentration of your
isolated plasmid by spectrophotometry at 260nm and its purify at 280nm (260/280 ratio).
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