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Detection of DNA Fragmentation: Toxicology Lab

This document provides a 9-step procedure for detecting DNA fragmentation using a DNeasy spin column. Key steps include lysing tissue in Buffer ATL and proteinase K, adding ethanol and centrifuging to bind DNA to the spin column membrane, washing with Buffers AW1 and AW2, and eluting purified DNA with Buffer AE. A second 4-step procedure is described for analyzing the isolated DNA on an agarose gel electrophoresis to qualitatively assess fragmentation.

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amaal ramadan
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23 views

Detection of DNA Fragmentation: Toxicology Lab

This document provides a 9-step procedure for detecting DNA fragmentation using a DNeasy spin column. Key steps include lysing tissue in Buffer ATL and proteinase K, adding ethanol and centrifuging to bind DNA to the spin column membrane, washing with Buffers AW1 and AW2, and eluting purified DNA with Buffer AE. A second 4-step procedure is described for analyzing the isolated DNA on an agarose gel electrophoresis to qualitatively assess fragmentation.

Uploaded by

amaal ramadan
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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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.

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