Plasmid Linearization
Reagents:
10X buffer
NotI
SalI
ddH2O
Agarose
Phenol
Chloroform
Ethanol
Glycogen
Sodium Acetate
Linear Acrylamide
Protocol:
1. Prepare 10 µg plasmid DNA in 5 µl ddH2O
2. Add:
a. 165 µl ddH2O
b. 20 µl NE Buffer #3
c. 10 µl NotI
3. Vortex, spin briefly
4. Incubate overnight at 37°C
5. Run 1% agarose gel:
a. 10 µl of reaction (digested plasmid)
b. 1 µl undigested plasmid
c. 1.6 µl 1kb ladder
d. 2.0 µl phi-x174 ladder
6. Continue if plasmid was cut completely
7. Do phenol/chlorofom extraction with 100 µl P/C pH 7.5
8. Pass top layer over G25 column
9. Add:
a. 1 µl glycogen
b. 30 µl 3M Sodium Acetate pH 7.5
c. 600 µl 100% Ethanol
10. Mix, precipitate for 20 minutes at -80°C
11. Spin 12 minutes at 14,000rpm
12. Wash pellet with 70% Ethanol
13. Wash pellet with 100% Ethanol
14. Dry pellet for 5 minutes
15. Dissolve pellet in 170 µl ddH2O
16. Add:
a. 20 µl NE Buffer for SalI
b. 10 µl SalI
17. Vortex, spin briefly
18. Incubate for 2 hours at 37°C
19. Run 1% agarose gel:
a. 6 µl of reaction (digested plasmid)
b. 1 µl undigested plasmid
c. 1 µl 1kb ladder
d. 1.4 µl phi-x174 ladder
20. Continue if plasmid was cut completely (2 bands)
21. Do phenol/chlorofom extraction with 100 µl P/C pH 7.5
22. Pass top layer over G25 column
23. Add:
a. 1 µl linear acrylamide
b. 30 µl 3M Sodium Acetate pH 7.5
c. 600 µl 100% Ethanol
24. Mix, precipitate for 20 minutes at -80°C
25. Wash pellet with 70% Ethanol
26. Wash pellet with 100% Ethanol
27. Dry pellet for 5 minutes
28. Dissolve pellet in 11 µl ddH2O
Svetlana Mikheeva, 13 August 1999