This
protocol is designed to purify single- or double-stranded DNA fragments
from
PCR. Fragments ranging from 100 bp to 10 kb are purified from primers,
nucleotides, polymerases, and salts using QIAquick spin columns in a
microcentrifuge.
Procedure
1. Add 5
volumes of Buffer PBI to 1 volume of the PCR sample and mix. For
example, add
500
µl of
Buffer PBI to 100
µl PCR
sample.
2. Place
a QIAquick spin column in a provided 2 ml collection tube.
3. To
bind DNA, apply the sample to the QIAquick column and centrifuge for
30–60 s.
4.
Discard flow-through. Place the QIAquick column back into the same
tube.
Collection tubes are re-used to reduce plastic waste.
5. To
wash, add 750
µl Buffer
PE to the QIAquick column
and centrifuge for 30–60 s.
6.
Discard flow-through and place the QIAquick column back in the same
tube.
Centrifuge the column for an additional 1 min.
7. Place
QIAquick column in a clean 1.5 ml microcentrifuge tube.
8. To
elute DNA, add 30
µl of
sterile DI water or EB to the center of the
QIAquick membrane, let the column stand for 1 min and centrifuge the
column for
1 min.
Materials
required
200
and 1000
µl
pipette tips
Sterilized
DI water
1.5
mL microcentrifuge tubes
Equipment
necessary
200
µl
micropipetter
1000
µl
micropipetter
Benchtop
centrifuge capable of 20,000 x g