Méthode recommandée pour l'isolation et la purification d'ADN
Que faire avec vos clones d'ADNc quand vous les recevez
The clones have been streaked into LB agar containing their respective antibiotic. Please store them at 4°C (not in a freezer).
As soon as possible, restreak the clones onto the same medium and incubate overnight at 37°C to obtain single colonies
A selection of at least 10 single colonies should be picked and grown in LB broth containing the appropriate antibiotic + 8%glycerol for subsequent freezing at -70°C.
These frozen stocks can then be regrown for plasmid isolation and purification.
Streaking out for single colonies is essential in case of contamination or cross-contamination, or if modifications of the original clone have taken place as the library has been replicated. If there is a problem, the more single colonies you streak out, the more likely it is that you can retrieve the original construct. You can also isolate the original clone from a contaminated sample by following this procedure.
Plasmid DNA mini-prep preparation
- Streak out plasmid onto LB + ampicillin (or chloramphenicol) plate. Incubate overnight at 37ºC (amp.) or 30ºC (CM).
- Dispense 5ml LB medium into a 50ml sterile tube. Add appropriate antibiotic. Inoculate with a single colony and incubate overnight in shaking incubator 37º C [30ºC (CM)] /170 rpm.
- [If a glycerol stock is required, transfer aliquot of 140µl to a 1.5ml microtube. Add 40µl 80%glycerol. Mix and freeze.]
- Take 2ml of the plasmid broth, transfer to a 2ml microtube, and centrifuge 15 min 3000 rpm.
- Pour off supernatant and discard. Re-suspend pellet in 200µl GTE.
- Add 5µl RNAase A stock (10mg/ml). Incubate 10 min room temperature.
- Lysis: Add 400µl 0.2 M NaOH/1%SDS (freshly made). Mix by inversion. Place on ice for 5 min.
- Add 300µl 3M K Ac. Invert to mix. Place on ice for 10 min
- Centrifuge 10 min at 13000 rpm in microfuge. Decant supernatant into a 1.5ml microtube containing 1 ml cold ethanol, vortex briefly and leave to stand for 30 min on ice.
- Centrifuge 10 min at 13000 rpm in microfuge. Remove ethanol carefully (preferably by suction technique).
- Add 200µl cold 70%ethanol, vortex briefly, centrifuge 10 min at 13000 rpm in microfuge, remove ethanol carefully (as above).
- Air dry pellet. Add 50µl TE and leave to re-suspend overnight at 4°C.
|10 ml GTE:||0.5 ml 20%glucose
0.25 ml 1M Tris/HCl pH 7.5
0.2 ml 0.5M EDTA pH 8.0.
9.05 ml sterile distilled water
100 ml 0.2M NaOH/1%SDS=
|0.8 g NaOH
95ml sterile distilled water.
PEG purification of plasmid DNA
a) Add an equal volume of 20%PEG solution in 2.5M NaCl. Incubate at room temperaturefor 5-10 min. Spinfor 10-15 min. at 13,000 rpm.
b) Remove supernatant and rinse pellet with 70%ethanol. Dry the pellet. Resuspend in 20 µl sterile distilled water.
c) Remove 5 µl and add to 2 µl loading buffer. Run out with appropriate DNA marker on a 1.5%TBE agarose gel to check that DNA recovery is good.
d) The DNA is now ready for sequencing.
PCR purification method
Clones can be amplified by PCR using the appropriate flanking primers. PCR products should then be purified using spin columns or the sAP/exo1 (shrimp alkaline phosphatase/exonuclease1) method prior to sequencing.
Werle. E, Schneider. C, Renner. M, Volker.M and Fiehn. W. (1994) Convenient single-step, one tube purification of PCR products for direct sequencing. Nucl. Acids Res. 22: 4354-4355.
Hanke. M and Wink. M. (1994) Direct DNA sequencing of PCR-amplified vector inserts following enzymatic degradation of primer and dNTPs. BioTechniques 17: 858-860.