Mini-prep for MSc Students
This procedure should be done.
NaOH Extraction of Plasmids from E. coli
1. Grow strain in L broth + antibiotic overnight or to stationary phase in 5 ml volume
2. Pellet cells (Bactria) after quick centrifugation and resuspend the pellet in 200µl
Solution I mix
3. Let sit on ice for 10 min
4. Add 400µl solution II, mix very well but not shake
5. Let sit on ice for 5 min (until it become transparent which should be less than 5 min)
6. Add 300µl of Solution III (3 M KOAc)
7. Mix very well and keep on ice for ~30 min
8. Spin 10 -15 min at highest speed in eppendorf centrifuge or 10 min at 15K rpm in
Beckman in 15 ml glass corex tubes
9. Take 750µl (8, or 20ml) of supernatant and add 500µl (fill tube) isopropanol (cold)
10. Freeze at -70oC for ~20 min or leave it in -20 for o/n
11. Let thaw and then spin for 10-15 min at highest speed in eppendorf centrifuge
12. Wash pellet with 75% EtOH (there is no need for a respin at this point if the pellet is
at the bottom of the tube) and you can repeat this wash for two times.
13. Dry pellet.
14. Resuspend in TE 40 to 90µl incubate at 65 C for 15 min if you are not going to the
following procedure. It must be stored at -20
Optional – for improving the purity:
15. If you have not added RNase to the solution I you can add RNAse to 10µg/ml and
incubate at 37oC for 30 min
16. Phenol extract 1X with an equal volume as the TE
17. Remove the aqueous phase, place in fresh tube and remove residual phenol by 2X
ether extraction
18. EtOH precipitate the DNA by adding 1/10 volume of salts and 3 volumes of 100%
EtOH. Incubate at -20oC for 15 min and spin for 15 min in eppendorf centrifuge at 4oC.
Wash the DNA pellet with 75% EtOH and re-spin for 5 min.
19. Dry the DNA pellet and resuspend in TE
For large scale preparations, the following can be performed.
This will remove small size RNA fragments and other impurities so that banding in CsCl
in unnecessary.
20. Resuspend DNA pellet from step 18 in 1 ml of TE
21. Load onto the top of 4 ml of 1 M NaCl (1.17g of NaCl in 20 ml of TE) in a SW50.1
tube
22. Spin at 20oC for 6 hours at 40,000 rpm
23. Pour off supernatant and resuspend pellet without drying in TE. The pellet is clear
and
contact shaped.
24. Store at 4oC
Plamid prep stock solutions:
Solution I mix For 20 ml:
0.4 ml of 0.5 M EDTA, pH 8.0
0.5 ml of 1 M Tris-HCl, pH 8.0
10µg/ml of RNase A (10µl of a 100µg/ml stock)
100 mg lysozyme (optional and not for this procedure)
0.454 ml of 40% glucose (optional and not for this procedure)
18.65 ml dd-H2O
Solution II
0.2 M NaOH + 1% SDS (fresh made)
2N NaOH For 100 ml:
8.0 g of NaOH pellets
dd-H2O, mix and store in plastic bottle
20% SDS For 100 ml:
20.0 g of SDS and add H2O to 100 ml. Stir at "low" heat setting to dissolve
Solution III
Prepared by mixing 60 ml of 5 M KOAc +
28.5 ml acetic acid + 11.5 ml H2O; pH will be 4.8
TE For 30 ml:
300µl of 1 M Tris-HCl, pH 8.0
60µl 0.5 M EDTA, pH 8.0
Add H2O to 30 ml
0.5 M EDTA, pH 8.0 For 150 ml:
27.92 g of EDTA (MW=372.24)
Add H2O to 150 ml
pH to 8.0 with NaOH to dissolve the EDTA
Autoclave and store
1 M Tris, pH 8.0 For 400 ml:
48.46 g Tris base (MW=121.14)
Add H2O to 400 ml
pH to 8.0
Autoclave and store