Competency

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==Preparing Competent Cells==
 
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Due to limited success using the Calcium Chloride method, we also utilised the TSS method in our experiments.
 
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==Preparing competent cells – TSS Method==
 
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1. Inoculate one E.coli colony in 5ml of LB medium (liquid) over night.
 
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2. Take 1ml of the overnight culture and add it to 100ml of pre-warm LB liquid in a 1L flask.
 
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3. Incubate until the optical density is about 0.5 at 600 nm (~3 hrs).
 
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4. Split 100 ml culture into 25 ml aliqouts and incubate on ice for 10min. All the subsequent steps should be carried out at 4°C and cells should be kept on ice as much as possible.
 
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5. Centrifuge for 10min at 3500rpm (4°C).
 
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6. Remove supernatant carefully by pouring and pipetting of the reminder. Place on ice immediately.
 
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7. Resuspend each pellet in 2.5 ml of chilled TSS buffer.
 
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8. Add 100 µl to Eppendorf tubes on ice. Freeze those aliqouts in a dry ice/ethanol bath and store at -80°C.
 
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==Preparing competent cells – Calcium Chloride Method==
 
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<br>
 
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1. Inoculate one E.coli colony in 5ml of LB medium (liquid) over night.
 
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<br>
 
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2. Take 1ml of the overnight culture and add it to 100ml of pre-warm LB liquid in a 1L flask.
 
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3. Incubate until the optical density is about 0.5 at 600 nm (~3 hrs).
 
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4. Aseptically transfer 50ml into a Falcon tube (repeat that step) and store on ice for 10min to cool the cultures to 0°C.
 
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5. Centrifuge at 4000rpm for 10min at 4°C.
 
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6. Take off supernatant and leave the tubes in an inverted position for about one minute to drain the reminder.
 
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7. Resuspend the pellet in 10ml of ice-cold 0.1M CaCl2.
 
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8. Centrifuge at 4000rpm for 10min at 4°C.
 
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9. Take off supernatant and leave the tubes in an inverted position for about one minute to drain the reminder.
 
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10. Resuspend in 2ml of ice-cold CaCl2 (0.1M) for each 50ml of original culture.
 
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11. At this point the cells can be split into aliqouts of 200µl and be frozen at -70°C.
 
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Latest revision as of 16:20, 9 July 2009