Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
CONTRIBUTOR:
OVERVIEW:
PROCEDURE:
The Laboratory of Donald Rio at the University of California, Berkeley
Although other miniprep protocols have fewer manipulations, this protocol consistently yields clean, clonable, sequencable plasmid DNA. The most important change in this protocol is the addition of RNase A after Step #7. This allows for the fewest manipulations and therefore the least contamination with this virtually indestructible enzyme.
1. Inoculate a 3 ml LB culture with a single colony from transformed bacteria and grow overnight to saturation.
2. Fill a 1.5 ml microcentrifuge tube with 1.5 ml of the saturated bacterial culture and centrifuge at full speed in a microcentrifuge for 30 sec.
3. Remove the supernatant (see Hint #2). Resuspend the pellet in 100 μl of Solution 1.
4. Add 200 μl of Solution 2. Invert the tube to mix the contents (do NOT vortex) until the mixture begins to clear. Incubate for 5 min on ice.
5. Add 150 μl of Solution 3. Invert the tube to mix the contents (do NOT vortex). Incubate for 5 min on ice.
6. Centrifuge at full speed in a microcentrifuge for 5 min.
7. Pour or pipette the supernatant into a fresh tube (see Hint #3).
8. Add 2 μl of 10 mg/ml RNase A to the supernatant. Vortex the tube to mix the contents and incubate for 10 to 30 min at room temperature.
9. Add an equal volume (approximately 500 ul) of Phenol:Chloroform and vortex the tube. Centrifuge at full speed in a microcentrifuge for 5 min.
10. Pipette the upper, aqueous phase to a fresh tube.
11. Add 2 volumes (approximately 1 ml) of 100% Ethanol to the tube, vortex the sample and centrifuge at full speed in a microcentrifuge for 5 min at room temperature.
12. Pour off the supernatant and add 200 to 500 μl of 70% Ethanol to the tube and vortex the sample. Centrifuge at full speed in a microcentrifuge for 5 min at room temperature.
13. Remove the 70% Ethanol with a pipette or aspirator. Dry the DNA pellet in a speed vac concentrator (vacuum microcentrifuge) for 5 min.
14. Resuspend the pellet in 50 μl of TE Buffer.
SOLUTION:
TE Buffer
10 mM Tris
pH 8.0
1 mM EDTA 
70% (v/v) Ethanol
Phenol:Chloroform
25:24:1 Phenol:Chloroform:Isoamyl alcohol (CAUTION! see Hint #1)
10 mg/ml RNase A
Store at -20°C
Solution 3
Store at 4°C.
2 M Glacial Acetic Acid
pH 5.6
3 M Potassium Acetate
Solution 2
1% (w/v) SDS
The fresher the Solution 2, the cleaner the preparation.
0.2 M NaOH
Make up fresh
Solution1
50 mM Glucose
10 mM EDTA
25 mM Tris-HCl
pH 8.0
LB Medium
1ml/liter 1.0 M NaOH
5g/liter Yeast Extract
Autoclave
10g/liter Tryptone
5g/liter NaCl
REAGENTS AND CHEMICALS:
Glacial Acetic Acid
Potassium Acetate
Sodium Chloride
Isoamyl Alcohol
Tris
SDS
Chloroform
RNase A
Phenol
Yeast Extract
Tryptone
EDTA
Ethanol
Sodium Hydroxide
Glucose
PROTOCOL HINTS:
2. You can store the pellets at -20°C for up to a week if desired.
3. For a quicker and dirtier preparation, skip Steps #8 through #10. Positive subclones can be Phenol:Chloroform extracted and reprecipitated with 100% Ethanol later.