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| - | {{:Team:Aberdeen_Scotland/header2}}
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| - | ==Transformation==
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| - | The following are different transformation methods used as part of our project
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| - | {{:Team:Aberdeen_Scotland/break}}
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| - | ==Transformation of TSS competent cells==
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| - | 1. Thaw 100µl of TSS/cells on ice
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| - | <br>
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| - | 2. Add 1µl of biobrick DNA, pipette gently to mix(100pg – 1 ng of plasmid in a volume of 1-2µl)
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| - | <br>
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| - | 3. Incubate on ice for 30 minutes
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| - | <br>
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| - | 4. Add 0.9ml SOC at 4°C
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| - | <br>
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| - | 5. Incubate for 1 hour at 37°C in shaker
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| - | <br>
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| - | 6. Spread 100 µl onto agar plate, containing appropriate antibiotic (see note ii.)
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| - | <br>
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| - | 7. Centrifuge remainder of cells at 200rpm / 5 min in a microfuge
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| - | <br>
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| - | 8. Remove all except 50 µl of supernatant: resuspend gently with a pipette
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| - | <br>
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| - | 9. Plate on a fresh agar plate, containing antibiotic
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| - | <br>
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| - | 10. Invert plates and incubate overnight at 37°C
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| - | <br>
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| - | Notes;
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| - | i) Antibiotic concentrations in LB:<br>
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| - | Ampicillin - 100 µg ml-1<br>
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| - | Tetracycline - 10 µg ml-1 in ethanol<br>
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| - | Chloramphenicol - 30 µg ml-1 in ethanol<br>
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| - | Kanamycin - 30 µg ml-1 in dH2O<br>
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| - | ii) Antibiotic concentrations in LB-agar:<br>
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| - | Ampicillin - 100 µg ml-1<br>
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| - | Tetracycline - 5 µg ml-1 in ethanol<br>
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| - | Chloramphenicol - 15 µg ml-1 in ethanol<br>
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| - | Kanamycin - 30 µg ml-1 in dH2O+<br>
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| - | {{:Team:Aberdeen_Scotland/break}}
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| - | ==Transformation of Calcium Chloride competent cells==
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| - | 1. Inoculate one E.coli colony in 5ml of LB medium (liquid) over night.<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.<br>
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| - | 3. Incubate until the optical density is about 0.5 at 600 nm (~3 hrs).<br>
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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.<br>
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| - | 5. Centrifuge for 10min at 3500rpm (4°C).<br>
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| - | 6. Remove supernatant carefully by pouring and pipetting of the reminder. Place on ice immediately.<br>
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| - | 7. Resuspend each pellet in 2.5 ml of chilled TSS buffer.<br>
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| - | 8. Add 100 µl to Eppendorf tubes on ice. Freeze those aliqouts in a dry ice/ethanol bath and sotre at -80°C.<br>
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| - | {{:Team:Aberdeen_Scotland/footer}}
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