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| - | {{:Team:Aberdeen_Scotland/css}}
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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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| - | <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. 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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| - | <br>
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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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| - | {{:Team:Aberdeen_Scotland/break}}
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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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| - | <br>
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| - | 3. Incubate until the optical density is about 0.5 at 600 nm (~3 hrs).
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| - | <br>
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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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| - | <br>
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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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| - | <br>
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| - | 7. Resuspend the pellet in 10ml of ice-cold 0.1M CaCl2.
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| - | <br>
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| - | 8. Centrifuge at 4000rpm for 10min at 4°C.
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| - | <br>
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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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| - | <br>
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| - | 10. Resuspend in 2ml of ice-cold CaCl2 (0.1M) for each 50ml of original culture.
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| - | <br>
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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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| - | {{:Team:Aberdeen_Scotland/footer}}
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