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Team:ESBS-Strasbourg/Miniprep: plasmid amplification
From 2008.igem.org
(Difference between revisions)
(→b.1) Protocol of the A.P. Silber course) |
(→b.1) MiniPrepProtocol of the A.P. Sibler course) |
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Or if quality is not so important you may use the following methode | Or if quality is not so important you may use the following methode | ||
| - | ==b.1) MiniPrepProtocol of the A.P. | + | ==b.1) MiniPrepProtocol of the A.P. Sibler course== |
*Spin down 1.5 mL of the Overnight culture (1min at 10 000rpm) | *Spin down 1.5 mL of the Overnight culture (1min at 10 000rpm) | ||
| - | *Take off the | + | *Take off the supernatant leaving around 100 µL medium |
*Resuspend the cells by vortexing | *Resuspend the cells by vortexing | ||
*Add 300µL TENS; vortex 2-5 sec | *Add 300µL TENS; vortex 2-5 sec | ||
*Add 150µL NaACo(3M pH5)fastly; vortex 2-5 sec | *Add 150µL NaACo(3M pH5)fastly; vortex 2-5 sec | ||
*Spin down 5 min at max. speed | *Spin down 5 min at max. speed | ||
| - | * | + | *Pipette the supernatant in a new tube without getting contamination of the pellet |
*Add 900µL ice cold ethanol (100% -20°C) and vortex | *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 | *Spin down 5 min at max. speed, take note of the orientation of the tubes | ||
| - | *Take off the | + | *Take off the supernatant being careful to not detach the pellet |
| - | *Wash with 1 mL (25% TE and 75%EtOH); DO NOT RESOLVE THE | + | *Wash with 1 mL (25% TE and 75%EtOH); DO NOT RESOLVE THE PELLET |
*Spin 2 min at max. speed | *Spin 2 min at max. speed | ||
| - | *Take off the | + | *Take off the supernatant and let dry the pellet completely |
*Resovle in 40µL RB (0,6 mL TE + 10 µL 1% gélatine + 2 µL RNase à 10 mg/mL) | *Resovle in 40µL RB (0,6 mL TE + 10 µL 1% gélatine + 2 µL RNase à 10 mg/mL) | ||
<br> | <br> | ||
Latest revision as of 09:23, 18 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) MiniPrepProtocol of the A.P. Sibler course
- Spin down 1.5 mL of the Overnight culture (1min at 10 000rpm)
- Take off the supernatant 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
- Pipette the supernatant in a new tube without getting contamination of the pellet
- 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 supernatant being careful to not detach the pellet
- Wash with 1 mL (25% TE and 75%EtOH); DO NOT RESOLVE THE PELLET
- Spin 2 min at max. speed
- Take off the supernatant and let dry the pellet completely
- 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
