52
HANS GERD NOTHW ANG AND FRIEDHELM HILDEBRANDT
enzymatic reactions. The nucleic acid is finally recovered from the aqueous
phase by precipitation (Figure 1).
Materials
Reagents
• salt-saturated phenol (SS phenol)
• chloroform
• isoamyl alcohol
• 70o/o and lOOo/o ethanol, ice-cold
• 3M sodium acetate (pH 5.2)
• TE-buffer (pH 7.6) (10 mM Tris-HCl (pH7.6), 1 mM EDTA (pH8.0))
Supplies
• 1.5 ml sterilized polypropylene tubes
• microcentrifuge
• chemical hood
• spectrophotometer
Procedure
ln advance
Note: Phenol is highly corrosive and can cause severe burns. W ear gloves,
gogges and protective clothing. Allmanipulations should be carried out in a
chemical hood. Dispose of used phenol in a glass receptacle, do not pour
down the drain.
1a) Before use, check the color and pH of phenol. Pink color indicates the
presence of oxidation products, which will darnage nucleic acids. The
phenol must therefore be discarded. If the phenol is unbuffered or the
pHisstill <7.8, DNA will tend to partition into the organic phase. In this
case, prepare buffered phenol (Sambrock et al. 1989).
1b) Add 0.5 g of antioxidant 8-hydro:xyquinoline (final concentration 0.1 o/o)
to a 2-liter glass beaker containing a stir bar. Gently pour SOOml of liquified phenol or melted crystals of redistilled phenol (melt at 65°C in a
water bath) and 500 ml of 0.5 M Tris-HCl (pH 8.0) into the beaker.
HANS GERD NOTHW ANG AND FRIEDHELM HILDEBRANDT
enzymatic reactions. The nucleic acid is finally recovered from the aqueous
phase by precipitation (Figure 1).
Materials
Reagents
• salt-saturated phenol (SS phenol)
• chloroform
• isoamyl alcohol
• 70o/o and lOOo/o ethanol, ice-cold
• 3M sodium acetate (pH 5.2)
• TE-buffer (pH 7.6) (10 mM Tris-HCl (pH7.6), 1 mM EDTA (pH8.0))
Supplies
• 1.5 ml sterilized polypropylene tubes
• microcentrifuge
• chemical hood
• spectrophotometer
Procedure
ln advance
Note: Phenol is highly corrosive and can cause severe burns. W ear gloves,
gogges and protective clothing. Allmanipulations should be carried out in a
chemical hood. Dispose of used phenol in a glass receptacle, do not pour
down the drain.
1a) Before use, check the color and pH of phenol. Pink color indicates the
presence of oxidation products, which will darnage nucleic acids. The
phenol must therefore be discarded. If the phenol is unbuffered or the
pHisstill <7.8, DNA will tend to partition into the organic phase. In this
case, prepare buffered phenol (Sambrock et al. 1989).
1b) Add 0.5 g of antioxidant 8-hydro:xyquinoline (final concentration 0.1 o/o)
to a 2-liter glass beaker containing a stir bar. Gently pour SOOml of liquified phenol or melted crystals of redistilled phenol (melt at 65°C in a
water bath) and 500 ml of 0.5 M Tris-HCl (pH 8.0) into the beaker.