New Alkali-Plasmid Preparation (Large and Mini)
LB 200 ml Large pres (max 2mg)
Sol I. 20 ml 10 ml
Sol II. 40 ml 20 ml
Sol III. 30 ml 15 ml
(1)
1. Centrifuge and pellet down, 3500rpm for 15min.
2. Freeze and thaw.
3. Sol I (よく攪拌する）
3. Add lysozyme in Sol I (痕跡程度入れる） (よく攪拌する）
4. Sol II (強く攪拌してはいけない、ホストDNAが寸断されコンタミる）
5. Sol III (よく攪拌する）
6. Centrifuge at 3500rpm for 15 min
7. Recover Sup
8. Isopropanol precipitation and dry up
(2)
1. Disolve in TE (1-2ml)
2. 50 μl of RNaseA(10mg/ml) at 37°C for 30 min.
3. Phenol/chloroform and chloroform treatment and PEG precipitation (equal volume PEG/NaCl) at -20°for 30min
4. Centrifuge and wash with EtOH
5. Disolve the DNA in 0.9 ml of TE.
<Optional>
5. Disolve the DNA in 0.9 ml of DW.
6. Add 0.1 ml of 2M sodium acetate pH4.0.
7. Extract with acid phenol and add 1/5 vol of chloroform and repeat
8. EtOH precipitation.
Materials:
Solution I: 50 mM glucose, 10 mM EDTA and 25 mM Tris-HCl, pH 8.0
Solution II: 0.2 N NaOH, 1% SDS
Solution III: 3 M potassium acetate, 2 M acetic acid