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Team:Hawaii/Plasmid Prep
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=== Protocol=== | === Protocol=== | ||
| + | 1. Grow single colony of ''E. coli'' at 37C overnight in 5 ml LB w/ antibiotic selection. <br> | ||
| + | 2. Microcentrifuge 1.5 ml cells for 20 sec at 10,000g. Discard supernatant.<br> | ||
| + | 3. Resuspend pellet in 100 μl GTE solution.<br> | ||
| + | :* 50 mM glucose | ||
| + | :* 10 mM EDTA | ||
| + | :* 25 mM Tris-HCl (pH 8.0)<br> | ||
| + | 4. Let sit for 5 min. at room temperature.<br> | ||
| + | 5. Add 200 μl NaOH/SDS solution.<br> | ||
| + | :* 0.2 M NaOH | ||
| + | :* 1% SDS | ||
| + | 6. Mix by tapping tube.<br> | ||
| + | 7. Incubate on ice for 5 min.<br> | ||
| + | 8. Add 150 μl potassium acetate solution.<br> | ||
| + | 9. Invert a few times to mix.<br> | ||
| + | 10. Incubate on ice for 5 min.<br> | ||
| + | 11. Microcentrifuge for 3 min. at 10,000g.<br> | ||
| + | 12. Transfer supernatant to a new tube.<br> | ||
| + | 13. Add 0.8 ml 95% ethanol.<br> | ||
| + | 14. Incubate for 2 min. at room temperature.<br> | ||
| + | 15. Microcentrifuge for 1 min. at 10,000g at room temperature. Remove supernatant.<br> | ||
| + | 16. Wash pellet w/ 1 ml 70% ethanol. Aspirate to dry (dry in hood).<br> | ||
| + | 17. Resuspend pellet in 30 μl TE buffer.<br> | ||
| + | |||
| + | ''Reference: Short Protocols in Molecular Biology'' | ||
Revision as of 01:26, 17 June 2008
Protocol
1. Grow single colony of E. coli at 37C overnight in 5 ml LB w/ antibiotic selection.
2. Microcentrifuge 1.5 ml cells for 20 sec at 10,000g. Discard supernatant.
3. Resuspend pellet in 100 μl GTE solution.
- 50 mM glucose
- 10 mM EDTA
- 25 mM Tris-HCl (pH 8.0)
4. Let sit for 5 min. at room temperature.
5. Add 200 μl NaOH/SDS solution.
- 0.2 M NaOH
- 1% SDS
6. Mix by tapping tube.
7. Incubate on ice for 5 min.
8. Add 150 μl potassium acetate solution.
9. Invert a few times to mix.
10. Incubate on ice for 5 min.
11. Microcentrifuge for 3 min. at 10,000g.
12. Transfer supernatant to a new tube.
13. Add 0.8 ml 95% ethanol.
14. Incubate for 2 min. at room temperature.
15. Microcentrifuge for 1 min. at 10,000g at room temperature. Remove supernatant.
16. Wash pellet w/ 1 ml 70% ethanol. Aspirate to dry (dry in hood).
17. Resuspend pellet in 30 μl TE buffer.
Reference: Short Protocols in Molecular Biology
