John M. S. Bartlett.pdf - Bio-Nica.info

Extraction of Ancient DNA 59
Table 1
Extraction Parameters
DNA preservation state *
Intermediate
Low
Parameters High degradation degradation degradation
Time of EDTA incubation (96)–120 h 48 h 24 h
Temperature of EDTA 20°C (20)–30°C (20)–30°C
incubation
Time of proteinase (60)–90 min 90 min 60 min
K incubation
Extraction reagent Sodium perchlorate Phenol (sodium Phenol (sodium
perchlorate)
perchlorate)
Amount of sodium acetate 64–117 µL 64–117 µL 64–117 µL
solution (2 M) to to
(128–233 µL) (128–233 µL)
Additional purification Wizard Prep (Wizard Prep) (Wizard Prep)
* DNA preservation state was determined by PCR amplification success and reproducibility of amplification
results applying the basic protocol optimization was based upon (2,5,16).
The preservation state was defined as high degradation when specific products were detectable in up
to 35% of the amplifications, displaying a low degree of reproducibility (12%) in typing results, and low
degradation when specific products were detected in 70 to 90% of the amplificates, with a reproducibility
of 86–91%.
of silica-solution (Glasmilk , Dianova) is added and the mix is shaken again for 10 min
at room temperature.
11. Using a DNA extraction device (Gene Pure, Perkin–Elmer), the mix is removed by pressure
and the DNA–silica complex is collected on a filter membrane. Alternatively, the mixture
can be separated by centrifugation at 4500g for 10 min.
12. The DNA–silica complex is washed with 2.8 to 3.8 mL of ethanol (abs.) for 5 min at
room temperature. Then, the alcohol is removed by renewed filtration or centrifugation.
Co-extracted salts may be removed using ethane (80Y).
13. Using an automated DNA extractor, the silica-bound DNA is manually removed from
the filter membrane with 500 µL of ethanol (abs.) and transferred to a 2-mL reaction
tube for further processing.
14. The DNA-silica complex is pelleted by centrifugation for 4500g for 4 min (Centrifuge
5804, Eppendorf) and the ethanol is discarded.
15. The resulting pellet is air-dried for approx 30 min at room temperature, redissolved in 50 µL
of sterile water (Ampuwa ® , Fresenius), thermally eluted at 50°C for 5 min at constant
shaking (thermal shaker 5437, Eppendorf), and stored at –20°C for further processing.
4. Notes
1. In case the state of DNA preservation of the material worked on is known, please change
the parameters concerned according to Table 1.
2. The use of sodium perchlorate may result in an inhibition of DNA polymerase during the
amplification process and therefore requires a further cleaning of the extracted DNA. For
this purpose, the Wizard PCR Preps purification system (Promega) following a modified
protocol (12,14) may be used.