Maxiprep of plasmid DNA
1. Inoculate one single colony in 10 ml LB with appropriate antibiotics.
Incubate overnight at 37oC with shaking.
2. Add 1 ml overnight culture to 500 ml LB with appropriate antibiotics.
Incubate overnight at 37oC with shaking.
3. Spin down the cells at 4K rpm with J6B for 10 min.
4. Resuspend the pellet in 40 ml ice-cold GTE (50 mM glucose,
10 mM EDTA, 25 mM Tris pH8.0) by vortexing.
5. Add 80 ml freshly prepared lysis buffer (0.2 N NaOH, 1% SDS),
mix well immediately until it becomes viscous. Leave on ice for
5 min.
6. Add 40 ml ice-cold 3K5Ac (60 ml 5 M KAc + 11.5 ml glacial acetic
acid + 28.5 ml water), mix well and leave on ice for 10 min.
7. Centrifuge at 4K rpm for 5 min with J6B.
8. Filter the supernatant through gauze into a fresh centrifuge
bottle.
9. Add 0.6 vol. isopropanol (i.e. 96 ml). Centrifuge at 4K rpm
with J6B for 10 min. Wash the pellet with 30 ml 70% EtOH. Spin
again and remove all the EtOH. Resuspend the pellet in 6 ml TE.
10. Weigh 8 g CsCl into an universal bottle. Reset zero for the
balance. Transfer the dissolved DNA to the CsCl and add TE to
give 7.2 g.
11. Add 0.8 g EtBr solution (stock: 5 mg/ml) and mix well.
12. Spin for 5 min. at 2K rpm with Beckman TJ-6. Transfer the
supernatant to an ultracentrifuge tube and, if necessary, top
it up with CsCl solution (1 g/ml in TE).
13. Balance the tubes TOGETHER with the cap and the black plug
to within 0.05 g difference.
14. Put the tubes into either Ti7.5 or Ti7.0 rotor.
15. Spin for 20 hr at 25oC at 55K rpm.
16. Put the ultracentrifuge tube in rack and remove the plug carefully.
17. Insert a 18-gauge needle, attached to 5 ml syringe, from the
top to collect the lower supercoiled DNA band, and keep it in
dark.
18. Add equal vol. of isopropanol (top phase) saturated with CsCl
solution (bottom phase).
19. Mix well and discard the upper phase. Repeat until the DNA
solution (lower phase) no longer appears pink.
20. Add 3 vol. TDW, 2.5 vol. EtOH and mix well.
21. Spin at 4k rpm for 10 min with J6B.
22. Wash with 70% EtOH twice and dry the pellet in Speedvac.
23. Resuspend the DNA in 0.5 ml TNE. Add RNase to 0.2 mg/ml. Incubate
at 37oC for 1 hr.
24. Add 5 ul 20% SDS (i.e. to 0.2%) and proteinase K to 0.2 mg/ml.
Incubate for 2 hr or overnight at 55oC.
25. Phenol-chloroform extraction 2X.
26. Add 0.1 vol. 3 M NaAc (pH 5.2) and 2.5 vol. EtOH.
27. Spin at 13k rpm with microfuge for 10 min. and wash with 70%
EtOH 2X.
28. Dry with Speedvac and resuspend the pellet in 1 ml TE.
29. Quantitate the DNA by spectrophotometer by transferring 5
ul to 1 ml TE in quartz cuvette and read OD at 260 nm for DNA,
280 nm for protein and 270 nm for phenol. (OD 20 = 1 mg/ml DNA;
260/280 should be about 1.8).