DNA Purification
GeneJet gDNA Purification Kit
Isolates High Quality Genomic DNA
SAMPLE PREPARATION
1 First, the cell membranes need to be broken down to
release the cellular contents. For cultured cells,
centrifuge the sample at 250 xg for 5 minutes.
Discard the supernatant and then wash with PBS
before centrifuging again. Remove the supernatant
and the cell pellet is now ready to use.
CELL LYSIS
2 To release the DNA from the cellular structures, first resuspend
the cell pellet in 200 µL of Phosphate-buffered saline (PBS) and
add 200 µL of Lysis Buffer. This buffer contains detergents that
breaks down cell membranes and proteins. Add 20 µL of
proteinase K to degrade any remaining proteins. Vortex to mix
the samples before incubating at 56ºC for 10 minutes. Next, add
20 µL of RNase A and vortex before incubating at room
temperature for 10 minutes. RNase A degrades RNA, ensuring
that only DNA remains for purification. This is important for
downstream applications where RNA could interfere with results.
Finally, add 400 µL of 50% ethanol and vortex.
BINDING
3 Transfer the supernatant to a spin column and
centrifuge at 6000 xg for 1 minute. The spin column
contains a membrane that selectively binds DNA while
allowing other cellular components to pass through.
Discard the collection tube with the supernatant and
transfer the spin column to a second collection tube.
WASHING
4 To remove contaminants, add 500 µL of Wash
Buffer 1 and centrifuge for 1 minute at 8000 xg,
discarding the flow-through. Repeat with 500 µL
of Wash Buffer 2, centrifuge at 13,000 xg for 3
minutes and discard flow through. Complete
another 1 minute centrifuge at 13,000 xg to
remove residual wash buffer and ethanol. These
GeneJet gDNA Purification Kit
Isolates High Quality Genomic DNA
SAMPLE PREPARATION
1 First, the cell membranes need to be broken down to
release the cellular contents. For cultured cells,
centrifuge the sample at 250 xg for 5 minutes.
Discard the supernatant and then wash with PBS
before centrifuging again. Remove the supernatant
and the cell pellet is now ready to use.
CELL LYSIS
2 To release the DNA from the cellular structures, first resuspend
the cell pellet in 200 µL of Phosphate-buffered saline (PBS) and
add 200 µL of Lysis Buffer. This buffer contains detergents that
breaks down cell membranes and proteins. Add 20 µL of
proteinase K to degrade any remaining proteins. Vortex to mix
the samples before incubating at 56ºC for 10 minutes. Next, add
20 µL of RNase A and vortex before incubating at room
temperature for 10 minutes. RNase A degrades RNA, ensuring
that only DNA remains for purification. This is important for
downstream applications where RNA could interfere with results.
Finally, add 400 µL of 50% ethanol and vortex.
BINDING
3 Transfer the supernatant to a spin column and
centrifuge at 6000 xg for 1 minute. The spin column
contains a membrane that selectively binds DNA while
allowing other cellular components to pass through.
Discard the collection tube with the supernatant and
transfer the spin column to a second collection tube.
WASHING
4 To remove contaminants, add 500 µL of Wash
Buffer 1 and centrifuge for 1 minute at 8000 xg,
discarding the flow-through. Repeat with 500 µL
of Wash Buffer 2, centrifuge at 13,000 xg for 3
minutes and discard flow through. Complete
another 1 minute centrifuge at 13,000 xg to
remove residual wash buffer and ethanol. These
