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Light Regulation of Plant Growth and Development
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Protocols
Generation and Transformation of chemically-competent E. coli
Generation of competent cells
All aspects of this prep are performed under sterile conditions and with sterile reagents on ice, unless otherwise specified.
1. Streak a plate of LB, no selection, with DH5α cells from a glycerol stock. Incubate overnight.
2. Select a single colony and transfer to 3 ml LB, culture O/N at 37° C with vigorous shaking.
3. Transfer 3 ml to 250 ml LB at 37°C, monitor growth until the culture is between 0.6 and 0.8 OD.
4. Move cells to sterile bottle chill on ice for 10 min and spin down.
5. Discard media and resuspend in 100 ml the following "competence" buffer, place on ice for 10 min.
10 mM Hepes
15 mM CaCl2
50 mM MnCl2
250 mM KCl
Mix all except MnCl2, pH to 6.7, add MnCl2, then move to 4°C. 250 ml is enough for 2 sets of cells. Filter sterilize.
6. Spin down cells and resuspend in 18.6 ml competence buffer.
7. Add 1.4 ml of DMSO and aliquot to microcentrifuge tubes, flash freeze in liquid nitrogen and store at -80°C. 50ul aliquots are convenient, this prep makes 400 transformations.
Transformation
1. Thaw cells on ice, add 10-50 ng of ligated plasmid, let stand on ice for 20-30 min.
2. Heat shock for 30 sec at 42°C.
3. Return to ice for 1 min.
4. Add 1 ml of LB or SOC, incubate at 37°C for 20-60 min, depending on selectable marker.
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