Team:ESBS-Strasbourg/Miniprep: plasmid amplification

==a) Remarks==

{| class="prettytable" style="width:50%; height:50px" border="1" cellspacing="0"
|- class="hintergrundfarbe5"
|-
| '''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 <br>
*12 to 16h over night at 37°C, 200-250rpm <br>

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) 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)
<br>
*10µL may contain 1µg of plasmid


[[Team:ESBS-Strasbourg/Protocols|''Protocols'']]