Difference between revisions of "Team:Manchester/Collaborations"
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Revision as of 14:39, 7 October 2017
Collaborations
Introduction
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Summaries
Materials (consumables):
LB broth (Luria Bertrani medium = rich media to grow bacteria)
TSS buffer (to prepare chemically competent cells)
S.O.C. medium (helps obtain the maximal transformation efficiency)
LB agar (gel where bacteria can grow)
Chloramphenicol (CAL) at stock concentration 25mg/ml
Preparation of chemical competent cells:
- Inoculate DH5α cells into 50mL LB and incubate at 37°C
- Monitor growth every 30 min by measuring optical density at 600nm (OD600) until it reaches OD600 = 0.4-0.6
- Harvest cells and prepare using 'TSS competent E.coli protocol' shown below:
LB Agar plates preparation:
- Prepare LB containing chloramphenicol (CAL) (at 25μg/ml)
- Melt LB in microwave (defrost setting for 15 min)
- Cool LB by running cold water over
- Stock of 25mg/ml CAL → so add 400μl CAL to 400ml LB
- Pour plates (in fume hood) and allow to solidify
Chemical Transformation:
- Add 1μl of DNA to 50μl of competent cells, mix well and place on ice for at least 30 min
- Heat shock cells at 42°C for 30 seconds, followed by 2 min incubation on ice
- Add 450μl of SOC medium to the cells and incubate for 45 min at 37°C (to allow protein expression (particularly the antibiotic resistance gene)
- Plate and spread (glass spreader sterilised over a flame and in ethanol) 50, 100, and 200μl of the cells into the agar plates made previously
- Incubate at 37°C for 2 hours
Reflection
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Sed condimentum turpis ut aliquam pellentesque. Quisque consectetur quam lorem, ut varius elit mollis euismod. Fusce gravida orci metus, nec cursus dui pulvinar quis. Vivamus accumsan magna tortor, quis egestas magna faucibus in. Morbi enim sem, sollicitudin viverra viverra et, tempor sit amet justo. Quisque malesuada magna eu lorem egestas, quis molestie lectus sollicitudin. Donec varius magna vel eros faucibus, vel cursus eros condimentum. Donec justo lacus, tempus sed vestibulum quis, semper vitae nulla. Nam vel accumsan mi. Cras molestie, risus in hendrerit laoreet, sapien mauris ultricies enim, in fermentum ligula nibh a tellus. In vitae sollicitudin nibh. Praesent convallis consectetur vulputate. Vestibulum faucibus augue a pretium interdum. Phasellus eros felis, tincidunt eget odio a, imperdiet consequat ipsum. Duis bibendum mattis efficitur. Etiam auctor id elit at fringilla. Donec pretium, ex id pellentesque varius, risus lacus pellentesque tortor, non tincidunt eros libero vel mi. Morbi viverra tempor mattis. Curabitur mattis sed dolor in aliquet. Maecenas nec metus at nibh gravida commodo id vel dolor. Morbi commodo cursus lacus, et ullamcorper nibh lacinia in. Mauris lectus enim, sodales vel dolor eget, volutpat volutpat purus. Quisque vitae odio pellentesque, pulvinar lorem eu, congue ligula. Morbi magna sem, pharetra sed sagittis quis, aliquam sit amet purus. Aliquam eget est ut ligula facilisis gravida ut ut ante. Nullam quis venenatis nulla. Nam facilisis ex in ligula consequat posuere. Pellentesque id arcu mattis, gravida elit a, hendrerit ante. Integer mollis justo sed vestibulum vehicula.
