When screening plasmid clones, do NOT use commercial kits but this protocol. It is not longer and much less expensive. Taxpayers will be grateful.

When archiving your plasmid, or preparing it for sequencing, do NOT use this protocol. Commercial kits will give you cleaner preps. Taxpayers will again be grateful since you won’t waste your time and therefore their money.


Solution II (Prepare fresh although one week old is OK) :
H2O Millipore :    8.9 mL
NaOH 10N :        200 μl
SDS 10% :        1mL

Let’s do it :

Pour overnight culture of bacteria in an eppendorf
Spin 1min 13Krpm RoomTemp, aspirate and trash supernatant in bleach
Add 100 ul of solution I
Vortex to resuspend pellet
Add 200 ul of solution II
Vortex quickly
Add 150 ul of solution III, close the cap and shake several times by hand
Place 5 minutes on ice

Spin 3 minutes at 13Krpm RoomTemp
Prepare 2 (or 3 if you’ll do a restriction digest) series of tubes

Transfer supernatant (~500 ul) to a fresh tube
Add 500ul Phenol :Chloroform 1 :1 ###FUME HOOD ! ###
Vortex
Spin 2 minutes at 13Krpm RoomTemp
Transfer supernatant to fresh tube
Discard phenol wastes in dedicated containers
Add 1mL of 100% Ethanol pre-cooled at -20°C
Vortex
Let 5 minutes at RoomTemp
Spin 5 minutes at 13Krpm RoomTemp
Discard supernatant
Dry pellet shortly
Resuspend in 50ul of TE+RNase
Use 5ul for restriction digest in 50ul

Buffers and reagents :

   
Solution I
50mM Glucose 50mM
25mM Tris.Cl pH 8.0
10mM EDTA (pH 8.0)

Prepare in batches of ~100mL, autoclave and store at 4°C.

Solution II (Prepare fresh although one week old is OK) :
H2O Millipore :    8.9 mL
NaOH 10N :        200 μl
SDS 10% :        1mL

Solution III (store at 4°C)
5M potassium acetate:     60mL
glacial acetic acid:        11.5 mL ### FUME HOOD, PROTECTIONS###
H2O:                28.5 mL

Phenol:Chloroform 1:1
-20°C cold absolute ethanol