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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES
REFERENCE NO: AHC/1998/3/3.1/2.3 Appendix 1
TITLE: PREPARATION OF DNA FROM CELL (SMALL SCALE PHENOL/ CHLOROFORM METHOD)
This procedure describes the isolation of genomic DNA from relatively small cell samples, 2 to 5 x 106 cells, by extraction with phenol and chloroform, prior to DNA fingerprinting.
- Fill out a DNA preparation log for each batch of samples and print out four sets of labels.
- Take 2 to 5 x 106 cells into a sterile microtube for DNA isolation. The quantity of cells may be considered sufficient if the pellet occupies the equivalent of 10 to 20ml. Pellets exceeding this volume should be split into two tubes.
- Pellet cells by centrifugation (10,000 to 13,000rpm using a microfuge, 1 min), remove supernatant and resuspend pellet.
- Wash with 1ml 1 x SSC (1 in 20 dilution of 20 x SSC). Mix and centrifuge for 1 min in a microfuge at 10,000 to 13,000rpm. Remove supernatant.
- Add 350ml 0.2M sodium acetate (1 in 10 dilution of 2M sodium acetate pH7.0) and mix by inversion.
- Add 10ml Ribonuclease A (10mg/ml) and mix.
- Add 25ul 10% SDS (10% SDS w/v in distilled or deionised water at pH 7.2) to each tube and mix. Incubate at 35 to 39°C for 30 min.
- Add 20ml Proteinase K (20mg/ml) and mix.
- Incubate at 55 to 60oC for 2 to 3 hrs. Alternatively, incubate at 56°C for up to 1 hr, then overnight in a water bath at 35 to 39oC.
- If the cell lysate is very viscous dilute 1 in 2 in 0.2M sodium acetate (see 5) and double volumes at subsequent steps. Add 200ml phenol/chloroform. Mix by inversion for approximately 1 min, until liquid appears milky.
- Microfuge at 10,000 to 13000rpm for 5 min. Remove the upper aqueous phase into a clean and pre-labelled microtube.
- Repeat steps 10 and 11 with this aqueous phase.
- Add 200ml chloroform/isoamyl alcohol.
- Microfuge as in step 11. Remove aqueous phase into a clean pre-labelled micro tube.
- Add 2 volumes (800ml) of absolute alcohol and invert tube several times to precipitate DNA.
- Microfuge for 1 minute (10,000 to 13,000rpm, microfuge), remove alcohol.
- Add 1ml 80% alcohol and mix.
- Microfuge as in step 16, remove all alcohol.
- Dry the pellet at 35 to 40oC for at least 30 to 60 min or 5 to 10 min under an infra-red lamp (minimum distance 10cm).
- Re-dissolve pellets in 200ml 0.2M sodium acetate (pH 7.5) at 55 to 60oC. Reduce volume to 100ml if the pellet is barely visible.
- Precipitate DNA with two volumes of absolute alcohol and microfuge at 10,000 to 13,000rpm for 2min.
- Aspirate alcohol and dry pellet as in step 19.
- Add 32ml of TE (10mM Tris, 1mM EDTA pH7.5 in deionised or distilled water) to the microtube containing the DNA pellet.
- Incubate at 55 to 60oC for 1 to 2 hours until the DNA has completely redissolved.
- If the DNA does not dissolve readily, leave at 2 to 8oC overnight and repeat step 24.
Quantify DNA using protocol AHC/1998/3/3.1/2.3 Appendix 11, prior to storage or setting up DNA digests (AHC/1998/3/3.1/2.3 Appendix 2).
Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
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