Apoptotic DNA Ladder Isolation Kit

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Seed 2x10^6/mL cells / well

Upstream tips
Seed 2x10^6/mL cells / well

Publication protocol

DNA Ladder Detection Protocol:
1. Induce apoptosis in cells by desired method. Concurrently incubate a control culture
without induction.
2. Wash cells with PBS (not provided) and pellet 2 x 106 cells by centrifugation for 5 min
at 500 x g. Carefully remove supernatant using pipette.
For adherent cells, gently trypsinize cells and then pellet cells.
For tissue samples, cut 50 mg tissues into very fine pieces or homogenize tissues in
PBS to generate cell suspension (Note: do not sonicate). Centrifuge to collect cell
pellet.
Note: The kit can detect DNA ladder from 105 apoptotic cells (100% apoptosis).
However, if the level of apoptosis in your sample is low, you can increase the cell
number up to 107. If using more than 2 x 106 cells per assay, you should proportionally
increase the volume of all reagents.
3. Extract the cell pellet with 50 µl DNA Ladder Extraction Buffer for 10 seconds at room
temperature with gentle pipetting. Centrifuge for 5 min at 1600 x g (~4500 rpm).
Transfer the supernatant to a fresh tube.
4. Extract the pellet again by repeating step 3. Combine the supernatant.
5. Add 5 µl Enzyme A Solution into the supernatant, mix by gentle vortex and incubate at
37°C for 10 min. (Note: If cells contain high level of DNase, then the incubation step
should be skipped, as high level DNase can digest DNA ladder generating smear
pattern.)
6. Add 5 µl Enzyme B Solution into each sample and incubate at 50 °C for 30 min or
longer (overnight is ok).
7. Add 5 µl Ammonium Acetate Solution to each sample and mix well. Add 100 µl
isopropanol (not provided), mix well, and keep at –20°C for 10 minutes.
8. Centrifuge the sample at maximum speed (~16K x g) for 10 minutes to precipitate
DNA. (Note: Microcentrifuges typically generate ~ 16K x g at 13K x rpm)
9. Remove supernatant, wash the DNA pellet with 0.5 ml 70% ethanol, centrifuge again
at maximum speed (~16K x g) to remove trace ethanol, and air dry for 10 minutes at
room temperature.
10. Dissolve the DNA pellet in 30 µl DNA Suspension Buffer (Note: No other loading
buffer needed for loading to the gel).
11. Load 15-30 µl of the sample onto a 1.2% agarose gel containing 0.5 µg/ml ethidium
bromide in both gel and running buffer.
12. Run the gel at 5 V/cm for 1-2 hours or until the yellow dye (included in the
suspension buffer) run to the edge of the gel.
13. Ethidium bromide-stained DNA can be visualized by trans-illumination with uv light
and photographed.

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