Reagents:
#1. Solution 1*
Reagent [final] [stock] volume, stock
Tris-Cl, pH 8.0 50 mM 1 M 25 ml
EDTA 50 mM 0.5 M 50 ml
sucrose 15% dry 75 grams
dH2O _______
500 ml
#2. Solution 2**
Reagent [final] [stock] volume, stock
dH2O 8 ml
NaOH 0.2 M 2 M 1 ml
SDS 1% 10 % 1 ml
10 ml
#3. Solution 3
Reagent [final] [stock] volume, stock
Potassium acetate 5 M 240 ml
glacial acetic acid 46 ml
H2O 114 ml
400 ml
#4. RNase (10 mg/ ml)
*Sterilized by autoclaving and best stored at 4 C
**Made fresh by addition of the indicated reagents in the indicated order.
Procedure
1. Grow a 2-3 ml overnight culture of bacteria for lysis (12 ml polypropylene tubes are convenient).
2. Transfer 1.5 ml to a microfuge tube and save the remainder up to several days at 4 C.
3. Spin down the bacteria at14000 rpm for 2 minutes
4. Resuspend the bacterial pellet in 100 µl Solusion I.
5. Add 200 µl of Solution 2 to the suspension and mix by inverting several
times.
6. Allow lysis to occur at room temperature for 5 minutes with occasional mixing.
7. Add 150 µl of solution 3 and mix by inverting several times.
8. Incubate at least 15 minutes on ice.
9. Centrifuge at 14K rpm at 4 C for10 minutes.
10. Carefully remove the supernatant with P1000, avoiding the loose pellet as much as possible, and place supernate in a fresh microfuge tube.
11. Add 5 ul of 10 mg/ml Rnase A and Incubate at 37 C for 15 minutes.
12. Add equal volume of Phenol/chloroform (1:1 ) and votex well.
13. Spin at 14k rpm for 5 minutes.
14. Transfer the supernate to a new tube.
15. Add 0.7 volume of Isopropanol and mix well by votex.
16. Spin DNA down at 14K rpm at RT for 10 minutes.
17. Wash DNA once with 70% ETOH and dry DNA in the speed/Vacum.
18. Resuspend DNA in 30 ul of H2O
19. Take 3 ul for digestion or 3 to 8 ul for sequencing.