Table T6. Procedure for DNA extractions from gabbro samples, Leg 205.

Table T6. Procedure for DNA extractions from gabbro samples, Leg 205.


Reagent		Preparation		Storage
2x buffer A with EDTA	NaCl	200 mM	1.16 g/100 mL	4°C
	Tris	200 mM	2.42 g/100 mL
	Na Citrate	2 mM	0.059 g/100 mL
	CaCl₂	10 mM	0.147 g/100 mL
	EDTA	50 mM
	Titrate to pH 8.0 with HCl, autoclave.
Poly A (10 mg/mL)	Make in 1.5-mL tubes with autoclaved water			-20°C
Pyrophosphate (10%)	10% sodium pyrophosphate, filter sterilize			Room temperature
Lysozyme (100 mg/mL)	Make in 1.5-mL tubes with autoclaved water			Make fresh
SDS 20%	Make with autoclaved water			Room temperature
Proteinase K (20 mg/mL)	Make in 1.5-mL tubes with autoclaved water			Fresh or -20°C
Phenol:chloroform: isoamyl-alcohol (24:24:1)	Saturate with pH 8.0 Tris			4°C
100% ethanol				Room temperature
3-M Na acetate	Titrate to pH 5.2 with acetic acid, autoclave			4°C
70% ethanol	Make with autoclaved water			4°C
TE buffer	10-mM Tris Cl, 1-mM EDTA; pH 8.0
Method
Lysis:
1) Thaw the sample enough to remove a 0.5- to 1.0-g aliquot of sample (1 mL of liquid sample) into 1.5-mL tubes. Keep the tubes on ice. If it is a liquid sample, you may want to wash the sample initially, especially if it is something like AMD (i.e., pellet cells at 12,000 g for 5 min at 4°C), and then decant supernatant. Resuspend cells in 2x buffer A then pellet cells again and decant supernatant.
2) Add 500 µL of 2x buffer A, 20 µL of poly A, 20 µL of 10% pyrophosphate, and 30 µL of lysozyme in that order (~5 mg/mL concentration lysozyme). Mix by gentle inversion. Incubate for 40 min at 37°C. Poly A and pyrophosphate will coat surfaces to prevent sorption of nucleic acids.
3) Add 10 µL of 20% SDS and 60 µL of proteinase K. (Only 0.3% of the final concentration of SDS is added at this step to prevent inhibition of proteinase K; the approximate proteinase K concentration = 2 mg/mL.) Mix by gentle inversion. Incubate for 30 min at 50°C.
4) Add 200 µL of 20% SDS (~5% concentration) and conduct three cycles of freeze-thaw by placing tubes in a dry-ice ethanol bath for 3 min at -70°C, then in a 65°C water bath for 5 min.
Extraction and precipitation:
5) Add 1 volume (~700 µL) of phenol:CHCl₃:IAA (24:24:1) and vortex to mix. Then spin at ~12,000 g at 4°C for 3 min to deposit sample debris. Pipette the aqueous supernatant (~600 µL of liquid) into a fresh 1.5-mL tube.
6) Extract samples with 1 volume phenol:CHCl₃:IAA (24:24:1). Mix by inversion and spin at ~12,000 g for 2 min at room temperature. Remove aqueous upper layer and put in new tube.
7) Precipitate nucleic acids by adding an equal volume (~500 µL) of ethanol and 0.1 volume (~50 µL) 3-M Na acetate, pH 5.2. Put on ice for 20 min to overnight then spin at ~12,000 g for 20 min at 4°C.
8) Decant supernatant; the pellet should be visible and colored, depending on the sample. Rinse pellets with 500 µL of cold 70% ethanol. Invert tubes on KimWipes and air-dry for ~30 min. Resuspend pellets in 2-30 µL of TE.
Purification:
9) Combine the DNA from 2 to 4 extracts. Purify the DNA through Clonetech Chroma Spin+TE100 columns. These can take ~70-200 µL and up to 100 ng of DNA.
10) Load 5 µL of purified DNA with 2 µL of GLD onto 1% agarose gel. Also, load into separate wells: MWmarker III (4 µL) and 50 ng and 100 ng of Lamda DNA. Run gel at 70 V for 30-60 min.
11) Observe bands of chromosomal DNA in the samples; these should run with or slightly above the 21-kbp band of the marker. Estimate DNA concentration in sample extracts. If necessary, DNA can be concentrated by precipitation (see steps 7 and 8 above).
12) If further purification is required, it can be done by gel purification.

Notes: DNA = deoxyribonucleic acid. EDTA = ethylenediamine tetraacetic acid. SDS = sodium dodecyl sulfate. TE = Tris EDTA. AMD = Acid Mine Drainage. IAA = isoamyl alcohol. GLD = gel loading dye.