Pharmacology and Toxicology

department

Detection of DNA
Fragmentation
Procedure

Toxicology Lab
Winter 2012
1- To 25mg of the tissue homogenate, add 180 μl Buffer ATL.
• 2-Add 20 μl proteinase K, mix by vortexing, and
Incubate at 55°C until the tissue is completely lysed (45 min.)

3-Vortex for 15 s. Add 200 μl Buffer AL to the sample, mix thoroughly

by vortexing, and incubate at 70°C for 10 min.
• 4-Add 200 μl absolute ethanol (96–100%) to the sample, and mix
thoroughly by vortexing.

5-Pipet the mixture from step 4 into the DNeasy spin column placed in
a 2 ml collection tube (provided). Centrifuge at ≥6000 x g (8000 rpm)
for 1 min.
Discard the filtrate in the collection tube

6- Place the DNeasy spin column in a new 2 ml collection tube

(provided), add 500 μl Buffer AW1, and centrifuge for 1 min at ≥6000 x
g (8000 rpm).
Discard the filtrate in the collection tube
7-Place the DNeasy spin column in a 2 ml collection tube (provided), add
500 μl Buffer AW2, and centrifuge for 3 min at full speed (14000 rpm) to
dry the DNeasy membrane.
Discard the filtrate in the collection tube

8-Following the centrifugation step, remove the DNeasy spin column

carefully so that the column does not come into contact with the flow-
through, since this will result in carryover of ethanol.

9-Place the DNeasy spin column in a clean 2 ml eppindorff , and pipette

200 μl Buffer AE directly onto the DNeasy membrane. Incubate at room
temperature for 1 min, and then centrifuge for 1 min at ≥6000 x g (8000
rpm) to elute.
1-Prepare a 1.5% agarose gel. Dissolve 1.5g agarose in 100 ml 1x
TBE.

2-Dissolve the agarose in a boiling water bath or in a

revolving-plate microwave oven. All the grains of
agarose should be dissolved and the solution totally
clear.

3-Add 2-3µl Ethidium bromide to the gel-buffer mix

(0.5µg/ml) and immediately pour the gel into the mold.

4-Insert a comb and allow the gel to cool before using ,

then carefully remove the comb and submerge the
casted (polymerized) gel in 1x TBE buffer.
Part II-Qualitative analysis of the isolated DNA by
Agarose Gel electrophoresis

Mix 10ul of DNA with 2ul loading dye.

Load 10ul per well.

Run the gel at 120V for 30-45 min.

Run samples 1/3 to 1/2 the length of the gel, without letting the
dye run off the bottom of the gel. Remove gel and visualize bands
under UV light.