Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
OVERVIEW: A small scale method of isolating plasmid DNA from recombinant DNA expressing bacterial cells.
PROCEDURE:
1. Centrifuge cells in a microcentrifuge at approximately 1,500 X g for 2 min.
2. Aspirate the supernatant and resuspend the cells in 50 μl of TE Suspension.
3. Add 100 μl of NaOH/SDS, mix thoroughly (see Hint #1) or vortex for a couple of seconds.
4. Add 75 μl of Acetate Solution and vortex for a couple of seconds.
5. Add 100 μl of Phenol:Chloroform solution and vortex for a couple of seconds.
6. At room temperature, centrifuge in a microcentrifuge at maximum speed for 2 min.
7. Save the aqueous phase (upper phase) and place in a new microcentrifuge tube.
8. Add 500 μl of 100% Ethanol, mix well by inversion and centrifuge in a microcentrifuge at maximum speed for 5 min.
9. Aspirate the supernatant without aspirating the DNA pellet (see Hint #2).
10. Allow the DNA pellet to air dry for a couple of minutes.
11. Resuspend the DNA in 50 μl of TE Buffer.
SOLUTION:
5 M Potassium Acetate
NaOH/SDS
1% (w/v) SDS
0.2 M NaOH
TE Suspension
10 mM EDTA
25 mM Tris
pH 8.0
TE Buffer
10 mM Tris
pH 8.0
1 mM EDTA
Phenol:Chloroform
1:1 Phenol:Chloroform
Acetate Solution
28.5 ddH2O
60 ml 5 M Potassium Acetate
11.5 ml Glacial Acetic Acid
REAGENTS AND CHEMICALS:
Sodium Hydroxide
Potassium Acetate
EDTA
Tris
Phenol
Acetic Acid
Ethanol
Chloroform
SDS
PROTOCOL HINT:
1. If you add each solution rapidly and forcefully in Steps #3, #4 and #5 you will not need to vortex the sample.
2. You can perform an additional alcohol wash (using 70% Ethanol) which should be performed exactly as the 100% Ethanol wash (Steps #8 and #9). You will not need the 70% Ethanol wash unless you will be using a very salt sensitive enzyme.