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Team:ESBS-Strasbourg/Miniprep: plasmid amplification
From 2008.igem.org
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| - | a) Remarks | + | ==a) Remarks== |
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| - | b) Protocol | + | ==b) Protocol == |
| - | + | *Take one colony and put it in 5 to 7mL of LB medium containing the antibiotic <br> | |
| - | + | *12 to 16h over night at 37°C, 200-250rpm <br> | |
| - | Notice that you will need about | + | Notice that you will need about 3mL of culture to perform the plasmid purification |
| + | |||
| + | You can either take the quick purification kit and follow the instruction of the kit discription. <br> | ||
| + | Or if quality is not so important you may use the following methode | ||
| + | |||
| + | ===b.1) Protocol of the A.P.-Silber course=== | ||
| + | |||
| + | *Spin down 1.5 mL of the Overnight culture (1min at 10 000rpm) | ||
| + | *Take off the supernagant leaving around 100 µL medium | ||
| + | *Resuspend the cells by vortexing | ||
| + | *Add 300µL TENS; vortex 2-5 sec | ||
| + | *Add 150µL NaACo(3M pH5)fastly; vortex 2-5 sec | ||
| + | *Spin down 5 min at max. speed | ||
| + | *Pipett the supernangant in a new tube without getting contamination of the pelett | ||
| + | *Add 900µL ice cold ethanol (100% -20°C) and vortex | ||
| + | *Spin down 5 min at max. speed, take note of the orientation of the tubes | ||
| + | *Take off the supernangant being careful to not detache the pellet | ||
| + | *Wash with 1 mL (25% TE and 75%EtOH); DO NOT RESOLVE THE PELETT | ||
| + | *Spin 2 min at max. speed | ||
| + | *Take off the supernangant and let dry the pelett completly | ||
| + | *Resovle in 40µL RB (0,6 mL TE + 10 µL 1% gélatine + 2 µL RNase à 10 mg/mL) | ||
| + | <br> | ||
| + | *10µL may contain 1µg of plasmid | ||
[[Team:ESBS-Strasbourg/Protocols|''Protocols'']] | [[Team:ESBS-Strasbourg/Protocols|''Protocols'']] | ||
Revision as of 09:28, 17 July 2008
a) Remarks
| Antibiotic | Working concentration |
| Ampicillin | 50-100µg/mL |
| Chloramphenicol | 25-170µg/mL |
| Kanamycin | 10-50µg/mL |
| Streptomycin | 10-50µg/mL |
| Tetracyclin | 10-50µg/mL |
b) Protocol
- Take one colony and put it in 5 to 7mL of LB medium containing the antibiotic
- 12 to 16h over night at 37°C, 200-250rpm
Notice that you will need about 3mL of culture to perform the plasmid purification
You can either take the quick purification kit and follow the instruction of the kit discription.
Or if quality is not so important you may use the following methode
b.1) Protocol of the A.P.-Silber course
- Spin down 1.5 mL of the Overnight culture (1min at 10 000rpm)
- Take off the supernagant leaving around 100 µL medium
- Resuspend the cells by vortexing
- Add 300µL TENS; vortex 2-5 sec
- Add 150µL NaACo(3M pH5)fastly; vortex 2-5 sec
- Spin down 5 min at max. speed
- Pipett the supernangant in a new tube without getting contamination of the pelett
- Add 900µL ice cold ethanol (100% -20°C) and vortex
- Spin down 5 min at max. speed, take note of the orientation of the tubes
- Take off the supernangant being careful to not detache the pellet
- Wash with 1 mL (25% TE and 75%EtOH); DO NOT RESOLVE THE PELETT
- Spin 2 min at max. speed
- Take off the supernangant and let dry the pelett completly
- Resovle in 40µL RB (0,6 mL TE + 10 µL 1% gélatine + 2 µL RNase à 10 mg/mL)
- 10µL may contain 1µg of plasmid
