I only obtain DNA smears from apoptosis induced cells, and no characterisitic DNA ladders/fragmentation
Inducing apoptosis in Jurkat cells as follows
Per 1 million cells in a well, final concentration:
- FasL (50ng/ml), 6 hours induction
- Camptothecin (2µM), 4 hours induction
I've tried the following protocols for isolating the fragmented DNA
- https://www.abcam.com/en-at/technical-resources/protocols/apoptosis-dna-fragmentation
Pellet cells by centrifugationLyse cells in 0.5 mL detergent buffer: 10 mM Tris (pH 7.4), 5 mM EDTA, 0.2% TritonVortexIncubate on ice for 30 minCentrifuge 27,000 x g for 30 minDivide supernatants into two 250 µL aliquotsAdd 50 µL ice-cold 5 M NaCl to each aliquot and vortexAdd ethanol and sodium-acetate and mix by pipetting up and down (600 µL ethanol and 150 µL 3 M sodium-acetate, pH 5.2.)Incubate tubes atat -80°C for 1 h.Centrifuge at 20,000 x g for 20 min; discard supernatants carefully.Pool DNA extracts together by re-dissolving the pellets in a total of 400 µL extraction buffer (10 mM Tris and 5 mM EDTA).Add 2µl RNase and incubate for 5 h at 37°CAdd 25 µL proteinase K (20 mg/mL) and 40 µL of buffer (100 mM Tris pH 8.0, 100 mM EDTA, 250 mM NaClIncubate overnight at 65°C.Extract DNA with phenol/chloroform/isoamyl alcohol (25:24:1) and precipitate with ethanolCarefully discard supernatantAir dry pelletResuspend in 20 µL Tris-acetate EDTA buffer supplemented with 2 µL of sample bufferElectrophoreseSimilar protocol by
- https://www.creative-bioarray.com/support/dna-laddering-assay.htm
As well as this protocol
Induce apoptosisPellet and resuspend all cells in 1ml PBS bufferAdd to chilled 70% EtOHOne set is incubated at RT for 72 hours, another set is incubated at -20 for 72 hoursCentrifuge at 1000g for 10 min at 4°CAspirate all the ethanolResuspend in 40µl phosphate-citrate buffer and transfer to EppieIncubate for 30 min at RTCentrifuge at 1000g for 5 minTransfer supernatant to new tubePlace in heating block at 60°c for 10-15 min to evaporate any residual ethanolAdd 3µl 0.25% Nonidet NP-40 (in dH2O); vortexAdd 3µl RNase A; vortex incubate, 30min at 37°CAdd 3µl Proteinase K; vortex, incubate 30min at 37°CAdd 2 µl 6x LD and load everything into a gelSee gel image for typical results.
WT is untreated control.
CPT is camptothecin treatment
Why am I not seeing the ladder/fragmentation characteristic of apoptosis in any of these protocols?
