BUFFER STOCK SOLUTIONS
EXTRACTION BUFFER STOCK
- 0.35 M sorbitol
- 0.1 M TrisHCl pH 8.0
- 5 mM EDTA pH 8.0
To make 500ml
- 31.9 g sorbitol
- 50 ml 1M TrisHCl pH 8.0
- 5 ml 0.5 M EDTA pH 8.0
- fill up to 500 ml MiliQ H2O
LYSIS BUFFER STOCK
- 0.2 M Tris HCl pH 8.0
- 0.05 M EDTA pH 8.00
- 2M NACl
- 2% CTAB
To make 500ml
- 100ml 1M Tri HCl pH 8.0
- 50 ml 0.5 M EDTA pH 8.0
- 200 ml 5 M NaCl
- 10 g CTAB
- fill up to 500 ml with MilliQ H20
SARCOSYL STOCK 5% (w/v)
FRESH BUFFER WORKING SOLUTION*:
0.5 % (w/v) sodiumdisulfite (= sodium metabisulfite)
2 % (w/v) PVP-40 (K29-32) Sigma
dissolve in required volume of extraction buffer stock; add same volume of lysis buffer stock and 0.4 volume of extraction (=lysis) buffer stock of sarcosyl stock.
For example to make 120 ml:
Add 0.6 g sodiumdisulfite (= sodium metabisulfite) and 2.4 g PVP-40 (K29-32) to 50 ml extraction buffer stock and dissolve; add 50 ml lysis buffer stock and 20 ml sarcosyl stock
For example to make 30 ml:
Add 0.15 g sodiumdisulfite (= sodium metabisulfite) and 0.6 g PVP-40 (K29-32) to 12.5 ml extraction buffer stock and dissolve; add 12.5 ml lysis buffer stock and 5 ml sarcosyl stock
*This buffer may settle into two layers on standing. Heat to 65ºC and shake immediately before adding to extraction tubes
PROTOCOL
For 15 ml Sarsted tubes:
- aliquot 6 ml of freshly prepared preheated to 65ºC well mixed “fresh buffer solution” and place tubes to the 65ºC incubator or water bath, (3, 4 days old “fresh buffer solution” works fine),
- grind required amount (same across all samples) of plant material in mortar and pestle under liquid nitrogen to fine powder,
- suspend powder in 6 ml “fresh buffer solution” kept at 65ºC (make sure there are no clumps, vortex if necessary),
- incubate at 65ºC for 1 h (can extend for another 30 min), invert tubes in every 20 minutes or incubate with gentle shaking,
- cool down for 5 min and add 6 ml of chloroform : isoamyl alcohol (24 : 1) mixture,
- mix well for 30 min,
- spin 20 min, 3000 x g, RT,
- transfer water phase to fresh tube, add same volume of ice cold isopropanol and invert tube ~ 10 times, nucleic acids should become visible,
- spin 30 min, 3000 x g, RT,
- discard supernatant, wash pellet with 2 ml 70 % EtOH,
- discard EtOH, dry pellet and dissolve in 200 µl – 500 µl 1 x TE (10 mM TrisHCl pH 8.0, 1 mM EDTA pH 8.0),
- check DNA quality and quantity on 0.8 % agarose gel. (If RNA quantity is several fold less than DNA, RNase treatment is not necessary for DArT applications).
For 2 ml Eppendorf tubes:
- aliquot 1 ml of freshly prepared preheated to 65ºC, well mixed “fresh buffer solution” and place
tubes to the 65ºC incubator or water bath, (3, 4 days old “fresh buffer solution” works fine), - grind required amount (same across all samples) of plant material in mortar and pestle under liquid nitrogen to fine powder,
- suspend powder in 1 ml “fresh buffer solution” kept at 65ºC (make sure there are no clumps, vortex if necessary),
- incubate at 65ºC for 1 h (can extend for another 30 min), invert tubes in every 20 minutes or incubate with gentle shaking,
- cool down for 5 min and add 1 ml of chloroform : isoamyl alcohol (24 : 1) mixture,
- mix well for 30 min,
- spin 20 min, 10000 x g, RT,
- transfer water phase to fresh tube, add same volume of ice cold isopropanol and invert tube ~ 10 times, nucleic acids should become visible,
- spin 30 min, 10000 x g, RT,
- discard supernatant, wash pellet with 2 ml 70 % EtOH,
- discard EtOH, dry pellet and dissolve in 250 µl of 1 x TE (10 mM TrisHCl pH 8.0, 1 mM EDTA pH 8.0),
- check DNA quality and quantity on 0.8 % agarose gel. (If RNA quantity is several fold less than DNA, RNase treatment is not necessary for DArT applications).
