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Team:IIT Delhi/Protocol
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| - | + | <b>Competent Cell Preparation</b><br> | |
| + | 1. Inoculate Escherichia coli DH5α culture in a test tube containing 5ml LB media and put on overnight incubation at 37⁰C. <br> | ||
| + | 2. Inoculate 1ml of the overnight culture in a 100ml flask for 2-3 hours to get an Absorbance of 0.4 to 0.6 so that cells reach the early log phase. <br> | ||
| + | 3. Centrifuge at 3000g for 10mins at 4⁰C.<br> | ||
| + | 4. Resuspend for 5 mins in 10 ml (for each oak ridge tube) 0.1M CaCl2 solution without vortexing.<br> | ||
| + | 5. After resuspension, keep on ice for 30mins.<br> | ||
| + | 6. Centrifuge as in (3).<br> | ||
| + | 7. Resuspend the whole pellet in 2-3ml 0.1M CaCl2 /15% Glycerol solution and dispense in 100uL aliquots.<br><br> | ||
| + | <hr style="width: 100%; height: 0.5px;"><br><br> | ||
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Revision as of 20:03, 27 September 2013
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| Protocols Competent Cell Preparation 1. Inoculate Escherichia coli DH5α culture in a test tube containing 5ml LB media and put on overnight incubation at 37⁰C. 2. Inoculate 1ml of the overnight culture in a 100ml flask for 2-3 hours to get an Absorbance of 0.4 to 0.6 so that cells reach the early log phase. 3. Centrifuge at 3000g for 10mins at 4⁰C. 4. Resuspend for 5 mins in 10 ml (for each oak ridge tube) 0.1M CaCl2 solution without vortexing. 5. After resuspension, keep on ice for 30mins. 6. Centrifuge as in (3). 7. Resuspend the whole pellet in 2-3ml 0.1M CaCl2 /15% Glycerol solution and dispense in 100uL aliquots. |
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