A detailed explanation of how to how to use the GeneJet purification kit and why each step is taken. Created by a graduate Biochemistry student who is now studying Biological Sciences at Cambridge University. This is designed to be used for all levels of learning, from GCSE to university. When lear...
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
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