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asr(½ð±Ò+2,VIP+0):thanks.
asr(½ð±Ò+2,VIP+0):thanks.
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6Â¥2008-01-10 18:37:50
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2Â¥2008-01-09 00:01:16
lihuanzheng
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reasonspare(½ð±Ò+2,VIP+0):¸Ðл°ïÖú
reasonspare(½ð±Ò+2,VIP+0):¸Ðл°ïÖú
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3Â¥2008-01-09 01:16:53
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asr(½ð±Ò+2,VIP+0):thanks.ÐÁ¿àÁË!
asr(½ð±Ò+2,VIP+0):thanks.ÐÁ¿àÁË!
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Prepare competent cell(1) 1. Pick a single colony of E.coli cells into 4~5 l LB at 37¡æ shaking 250 rpm overnight(15~16 h).(or 1:1000, E.coli : LB) for example: 5ul E.coli for 5 ml LB. 2. 100 ml +2ml overnight E.coli culture at 37¡æ shaking 2 h.(about OD=0.4) 3. Aliquot separate culure into two 50 ml prechilled sterile polypropylene tubes and leave the tube on ice 5~10 min. centrifuge cells 7 min 3000 rpm, 4¡æ. 4. Resuspend(gently) each pellet in 10 ml ice-cold 0.1M Cacl2 solution. Centrifuge 5 min at 3400 rpm(1100 g), 4¡æ. 5. Resuspend each pellet in 10 ml ice-cold 0.1M Cacl2 solution. Keep resuspended cells on ice for 30 min. centrifuge 5 min at 1100 g, 4¡æ. 6. Resuspend each pellet completely in 4 ml of ice-cold Cacl2 solution, Aliquot 200 ul into prechilled, sterile polypropylene tubes. Freeze immediately at 4¡æ(last two weeks for using). Or add glycerol at 15% final concentration, freeze immediately at -70¡æ(last several month for using). For example: 3 ml Cacl2-E.coli with 1 ml 50% glycerol. |
4Â¥2008-01-09 21:04:23














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