DNA测序的基本原理
来自 : 蚂蚁淘
Procedure - pick one colony
- inoculate in 3 ml of the appropriate media
- grow at 30° overnight
- pellet the cells (5 min, 5000g)
- wash 1X in sterile ddH2O
- re- suspend the pellet in 200 µl EthOH (optional: +2 µl PMSF)
- add approx. 100 µl glass beads (0.5 µ) in a reaction tube (you can use the cap of a 0.5 ml reaction tube as bucket)
- vortex vigorously for 2 min., cold (optimal is an auto- vortex like “vortex turbo-mix”)
- collect the supernatant in an fresh reaction tube
- add again 200 µl EthOH
- vortex
- collect the supernatant
- add again 200 µl EthOH
- vortex for 2 min
- (optional: repeat this again)
- collect the supernatant
- incubate at -20°C for 30 min or longer
- centrifuge 16000g, for 15 min at 4°C
- remove supernatant and re- suspend pellet in SDS page sample buffer and directly
- boil the samples to denature
- SDS Page and western blotting
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