Plasmid DNA Miniprep
- inoculate 2 ml medium with a single colony and grow overnight (12-24 h)
- spin 1.5 ml (1 min, full speed, microfuge), keep rest in refrigerator
- remove supernatant with a syringe
- resuspend pellet in 150 µl P1 (vortexing)
- add 150 µl P2, shake gently
- incubate for 5 min at RT
- add 150 µl P3, shake gently
- spin at RT (3 min, full speed, microfuge)
- pipet carefully 440 µl of supernatant into a new tube
- add 1 ml Ethanol (RT)
- spin at RT (5 min, full speed, microfuge)
- pour off supernatant
- wash pellet with 150 µl 70 % Ethanol
- dry pellet in speed vac
- dissolve in 40 µl
H2O
- use 2 - 4 µl for restriction analysis
- the DNA is suitable for all enzymatic reactions including sequencing
Buffers:
P1: 50 mM Tris/HCl pH 8.0; 10 mM EDTA;
RNAse A 100 µg/ml.
P2: 0.2 M NaOH; 1 % SDS.
P3: 2.55 M KOAc pH 4.8.
