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Competent Cells Preparation
Competent Cells Preparation
Reagents Preparation
CM1 (25 ml)
10 mM NaOAC pH 5.6 0.25 ml of 1M NaOAC stock
50 mM MnCl2 1.25 ml of 1M MnCl2 stock
5 mM NaCl 0.125 ml of 1M NaCl Stock
DW 23.375 ml
--> Filtered
CM2 (10 ml)
10 mM NaOAC pH 5.6 0.1 ml of 1M NaOAC stock
70 mM CaCl2 0.7 ml of 1M CaCl2 stock
5 mM MnCl2 0.05 ml of 1M MnCl2 stock
5% glycerol 0.5 ml of glycerol
DW 8.65 ml
--> Filtered
-----------------------------------------------------------
1. Streak frozen bacteria stock onto LB plate --> incubate overnight
2. Pick 1 colony and inoculate in 2 ml LB broth --> Shake @ 37 ◦C overnight
3. Transfer 1 ml into 100 ml LB broth --> Shake @ 37 ◦C for 2 3 hours until reaching the OD of 0.45 5
4. Transfer to 2 x 50 ml tube --> incubate on ice for 5 minutes --> centrifuge at 2700 rpm for 5 minutes at 4 ◦C
5. Discard supernatant and add CM1 10 ml / 50 ml culture (i.e. 20 ml total)
6. Incubate on ice for 45 minutes. Meanwhile, cool tubes in -80 ◦C freezer.
7. Centrifuge at 2700 rpm for 5 minutes at 4 ◦C
8. Add 1 ml CM2 / 50 ml culture (i.e. 2 ml total)
9. Aliquots 100 µl cells / tube --> Store at -80 ◦C
| Create Date : 28 มกราคม 2551 |
| Last Update : 28 มกราคม 2551 15:58:57 น. |
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If there is a stage at which an individual life becomes truly adult, it must be when one grasps the irony in its unfolding and accepts responsibility for a life lived in the midst of such paradox. One must live in the middle of contradiction, because if all contradiction were eliminated at once life would collapse. There are simply no answers to some of the great pressing questions. You continue to live them out, making your life a worthy expression of leaning into the light.
-Barry Lopez Arctic Dreams
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