Apoptotic DNA-Ladder Kit

This kit provides a way to isolate apoptotic DNA fragments for DNA ladder analysis. The purification method used is much faster than other DNA purification methods (e.g., phenol/chloroform extraction, DNA precipitation). Purified DNA can be mixed directly with gel loading buffer and analyzed on an agarose gel.

The study of cell death involves characterizing mortality as apoptotic or necrotic. Apoptosis can be characterized by:

• Prelytic, non-random fragmentation of DNA (“ladder” pattern after agarose-gel electrophoresis)1
• Formation of membrane-bound vesicles (or “apoptotic bodies”)
• Cell shrinkage due to a concentration of cytoplasm

Similarly, necrosis (or physiological cell death) is characterized by

• Random digestion of DNA (DNA smear after agarose-gel electrophoresis)
• Swollen organelles and cells, resulting from loss of membrane integrity and cell lysis
• Postlytic DNA fragmentation

As these differences indicate, the classification of cell death can be accomplished through observing cell morphology, or less subjectively, by analyzing genomic DNA. Resolving the DNA on a gel provides a quick and documentable form of data to differentiate between apoptosis and necrosis.

Only nucleic acid will bind to the glass fiber filters under the conditions outlined in the kit. Salts, proteins, and other cellular components do not bind.

The Apoptotic DNA-Ladder Kit provides rapid isolation of DNA, which can be analyzed and characterized by gel electrophoresis for the determination of apoptotic cell death.

1 kit containing 6 components.

Blood or cell lysis is accomplished by incubating the sample with the special Binding/Lysis Buffer. The sample is centrifuged through a column that contains glass fiber fleece. In the Binding/Lysis Buffer, nucleic acids quickly and preferentially bind to the surface of the glass fibers. The Washing Buffer rinses away salts, proteins, and other cellular debris. DNA is subsequently collected via a centrifugation step with elution buffer.

Working solution: Note: Before starting a purification reaction warm the Elution Buffer to 70 °C, all other reagents should be at 15 to 25 °C.

Preparation of Working Solutions

• Add 80 ml ethanol, analysis grade, to Washing Buffer
• Dissolve Positive Control (violet cap) in 400 μl Binding/Lysis Buffer and mix immediately.
• Incubate for 10 minutes at 15 to 25 °C.

Preparation of Working Solutions for DNA Gel Electrophoresis

EDTA solution (0.5 M)
Dissolve 18.6 g EDTA in 80 ml double dist. water and stir. Adjust pH 8.0 ± 0.1 with 1 M NaOH. EDTA solubilizes at alkaline pH only. After solubilization fill up to 100 ml with double dist. water.

TBE-Buffer
Dissolve 5.4 g Tris, 2.8 g Boric acid in 800 ml double dist. water and add 2 ml
of 0.5 M EDTA solution . Stir until dissolved, final pH 8.0 ± 0.1. Fill up to 1 liter with double dist. water.

Ethidium bromide stock solution
Dissolve 50 mg ethidium bromide in 5 ml double dist. water (ethidium bromide is a mutagen and potential carcinogen; gloves should be worn and care should be taken when handling ethidium bromide solutions).
Alternatively SYBR Green I Nucleic Acid Gel Stain can be used instead of Ethidium bromide.

Loading Buffer (10x)
Dissolve 0.1 g sodium dodecyl sulfate, 25 mg Bromophenol Blue in 7 ml double dist. water and add 3 ml glycerol
Storage conditions (working solution): The positive control can be used for 14 days after preparation, when storing the isolated control DNA at -15 to -25 °C.

For life science research only. Not for use in diagnostic procedures.

NOTICE TO PURCHASER: This is a product licensed under patents owned by Qiagen.