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Team:Bonn/Protocols
From 2012.igem.org
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** add 250 µl LB medium of 37 °C | ** add 250 µl LB medium of 37 °C | ||
** incubate for 45 min at 37 °C, 800 rpm | ** incubate for 45 min at 37 °C, 800 rpm | ||
| + | |||
| + | === Transformation of Ligations (in DH5alpha or XL1Blue) === | ||
| + | * thaw bacteria on ice or carefully in your hand | ||
| + | * add 2-4µl Ligation mixture to 50µl bacteria | ||
| + | * incubate 30min on ice | ||
| + | * heat shock 30s-45s (XL1Blue preferably 35s) at 42°C | ||
| + | * incubate 6min on ice | ||
| + | * add 250µl prewarmed LB medium (37°C) | ||
| + | * incubate for 45min at 37°C, 800rpm | ||
| + | * plate 300µl on apropiate antibiotic | ||
| + | * dry 15min at room temperature | ||
| + | * incubate at 37°C over night | ||
Revision as of 22:53, 25 September 2012
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Contents |
Lab protocols
On this page you can find a list of important standard protocols we used in our project.
Preparation of chemocompetent DH5-alpha cells
- liquid cultures, OD_600 of 0,6-0,8
- centrifugation at 4 °C, 4500 g, 10 min
- decantation
- resuspend with 40 ml inoune transformation buffer
- repeat centrifugation, decantation and resuspendation
- centrifuge and decantate again
- resuspend in 20 ml inoune transformation buffer
- add 1,5 ml DMSO
- shock freeze in liquid nitrogen
Retransformation of BioBricks
- add 10 µl sterile dest water to DNA on plate
- incubate for 10 min
- take 2 µl, leave rest on plate
- store plates at -20 °C
- add the 2 µl DNA solution to 5 µl competent DH5-alpha
- incubate for 30 min on ice
- heat shock for 45 s at 42 °C
- incubate 3 min on ice
- add 250 µl LB medium of 37 °C
- incubate for 45 min at 37 °C, 800 rpm
Transformation of Ligations (in DH5alpha or XL1Blue)
- thaw bacteria on ice or carefully in your hand
- add 2-4µl Ligation mixture to 50µl bacteria
- incubate 30min on ice
- heat shock 30s-45s (XL1Blue preferably 35s) at 42°C
- incubate 6min on ice
- add 250µl prewarmed LB medium (37°C)
- incubate for 45min at 37°C, 800rpm
- plate 300µl on apropiate antibiotic
- dry 15min at room temperature
- incubate at 37°C over night
