From 2012.igem.org
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
Mini-Prep (Promega)
- add 1,5 ml cell culture in an eppi
- zentrifuge for 30 sec, max speed
- decantate
- resuspend with 600 µl dest water
- ad 100 µl cell lysis buffer
- after ca. 1 min add 350 µl of neutralization buffer
- centrifuge 3min maximum speed
- put Minicolumn into Collection Tube and transfer supernatant into PureYield^TM Minicolumn
- centrifuge for 15 sec at max speed
- centrifuge for 15 sec at max speed with 200 µl Endotoxin Removal Wash
- centrifuge for 30 sec at max speed with 400 µl Column Wash solution
- put Minicolum into new eppi, abolish liquid
- transfer 30 µl elution buffer into Minicolum, wait for 1 min
- centrifuge for 15 sec at max speed
- store DNA at -20 °C
Midi-Prep (Promega)
- centrifuge 50 ml of liquid cell culture for 10min at 5000g
- decantate
- resuspend with 3 ml resuspension solution
- add 3 ml cell lysis solution and incubate for maximal 3 min at room temperature
- add 5 ml neutralization solution
- centrifuge for 20 min at 20 °C, 5000g
- vacuum pump lysat through cleaning column into binding column
- abolish cleaning column
- vacuum pump with 10 ml endotoxin removal wash solution
- vacuum pump with 20 ml column wash solution
- dry membrane by vacuum
- dd 600 µl nuclease free water on membrane
- centrifuge for 5 min at 1750 g into eppi
Glycerol Stocks (iGEM)
- autoclave glycerol (60%)
- add 1,5 ml cell culture and 0,5 ml Glycerol in a kryo tube
- vortexe
- shock freeze in liquid nitrogen
- store at -80 °C
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