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now: Thu Jun 27 01:59:27 2019 ... mod: Wed Dec 9 11:39:00 2015
Oh, the all by mysewf phase where do our litlte people go? Oh, but Astrid is completely darling & looks so precious getting her first haircut the perfect mixture of sweet baby girl & independent big girl!And I totally hear you with being SO overwhelmed by the number of BIG blogs out there, I hardly find myself reading them much anymore. I prefer a blog where I feel some connection to the writer. Just too litlte time in life to get all too wrapped up in strangers lives, but it can totally suck you in! So I'm trying each & every day to make those in front of my my highest priority.Melanie H. recently posted..
Electrocompetent Cell Preparation
This protocol can be used to make E. coli
stocks that transform at high efficiency by BacterialElectroporation
- StreakForIsolation onto an agar plate.
- pick a single colony from the isolation plate and innoculate into 3 ml medium. grow overnight
- use LB or high-nutrient medium such as Circlegrow
- add the entire 3 ml culture to 500 ml prewarmed medium and incubate with shaking until OD600 is 0.5 to 0.7
- do not let the culture overgrow
- 0.5 to 0.6 works best
- if using a baffled flask for improved culture aeration, you can use 1000 ml of prewarmed medium instead of 500 ml
- chill culture with shaking in a water / ice slurry to stop growth
- from this point forward, the cells must be kept at 4C or on ice at all times and all tubes, bottles and solutions must be pre-chilled to 4C
- centrifuge culture to pellet cells
- 6000g for 10 minutes works well. eg: 250 ml bottle, Sorvall GSA rotor, 6000 rpm
- if the size of the culture required use of multiple centrifuge tubes to get the cells to pellet, the pellets should all be combined together into a single tube for all of the subsequent processing steps
- resuspend cells in 200 ml ice-cold sterile water and centrifuge to pellet
- resuspend cells in 50 ml ice-cold sterile 10% glycerol and centrifuge to pellet
- eg: 50 ml oakridge tube, Sorvall SS34 rotor at 7000 rpm or SA600 rotor at 6500 rpm
- remove supernatant and estimate volume of the cell pellet
- resuspend cells in an equal volume of ice-cold sterile 10% glycerol
- aliquot into prechilled tubes and freeze on crushed dry ice
- do not freeze in liquid nitrogen
- do not add ethanol to the crushed dry ice
- 50 μl is a good aliquot size
- store at -80C
- important control: take one of the newly prepared compentent cell aliquots and spread untransfected on both ampicillin and kanamycin plates. incubate overnight at 37C. there should be no colonies on these plates. any colonies will indicate contamination with drug resistant bacteria -- discard this compenent cell preparation and try again
- this kind of contamination really can happen so do not skip this step
- filter sterilization of solutions is simple and effective
- as the salts are rinsed out of the cells, the cell pellet will become very prone to spontaneous dispersion following centrifugation. Combine cells as possible into a single centrifuge tube and carefully decant the rinses immediately following centrifugation to minimize cell losses.