Transformation

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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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==Transformation of TSS competent cells==
 
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1. Thaw 100µl of TSS/cells on ice
 
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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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3. Incubate on ice for 30 minutes
 
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4. Add 0.9ml SOC at 4°C
 
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5. Incubate for 1 hour at 37°C in shaker
 
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6. Spread 100 µl onto agar plate, containing appropriate antibiotic (see note ii.)
 
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7. Centrifuge remainder of cells at 200rpm / 5 min in a microfuge
 
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8. Remove all except 50 µl of supernatant: resuspend gently with a pipette
 
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9. Plate on a fresh agar plate, containing antibiotic
 
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10. Invert plates and incubate overnight at 37°C
 
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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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==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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Latest revision as of 16:21, 9 July 2009