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DNA Extraction of Ancient Bone

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Brighton Troha

on 4 October 2013

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Transcript of DNA Extraction of Ancient Bone

DNA Extraction of Ancient Bone
Preparation
Steps three and four
After phase separation by centrifugation at room temperature at 12000 r.p.m. for 10 min

250 µl supernatant was transferred to a 1.5 ml Eppendorf tube and 3.5 µl 1 µg/µl Dextran Blue , 250 µl 4 M NH4-acetate and 500 µl 96% EtOH were added and mixed by vortexing.
Steps five and six

The DNA is precipitated at –70°C for 7 min and centrifuged at 14000 r.p.m. at 4°C for 15 min.

The pellet is redissolved in 20–30 µl deionised, distilled water.

The remaining extract is stored at –20°C.
Dextran Blue
EDTA
Prohibits degredation.
Protects the DNA from digestion by nucleases. Ions form covalent bonds with the divalent cations and prevent them from reacting with nucleases. As a result, the enzymes are deactivated and will no longer cause a threat to the DNA.
Procedure
Physically powdered bone (750 mg) was suspended in 1.6 ml extraction buffer (0.1 M EDTA, 0.5% N-laurylsarcosine-Na salt, 100 mg/ml proteinase K)

Vortexed and incubated overnight at 37°C with continuous vertical rotation.
Surface material was removed from the bones by washing with diluted bleach and distilled water.
A 2 × 5 cm portion section was cut from each bone diaphysis, and the surfaces of these portions were removed (at least 2–3 mm deep) with a sand disk in order to get rid of modern DNA contamination.
The cleaned bone fragments were treated with UV light at 1.0 J/cm2 for 30 min, and mechanically ground into a fine meal in a sterile agate mortar.
Steps one and two

N-lauroylsarcosine-Na salt
Used for solubilization and separation of membrane proteins and glycoproteins.
Proteinase K
An enzyme used in order to digest proteins and remove contaminants from DNA

DNA is insoluable
DNA is insoluble in alcohols such as ethanol, it will aggregate together, resulting in a pellet upon centrifugation.
Dextran Blue effectively coprecipitates low concentrations of DNA and colours the pellet.

Ethanol
Works Cited
"Ancient DNA Extraction from Bones and Teeth." Nature.com. Nature Publishing Group, 12 July 2007. Web. 04 Oct. 2013.

"DNA Isolation from Eukaryotes." : DNA Isolation from Eukaryotes. N.p., n.d. Web. 04 Oct. 2013.

"Exploratorium : Evidence : Extracting DNA from Neanderthal Bones." Exploratorium : Evidence : Extracting DNA from Neanderthal Bones. Dr. Johannes Max Planck Institue for Evolutionary Anthropolgy, n.d. Web. 04 Oct. 2013.

Rice, George. "DNA Extraction." DNA Extraction. Montana State University, n.d. Web. 04 Oct. 2013.
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