Team:Newcastle University/Protocols

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==Protocols==
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[[Agarose Gel Electrophoresis]]
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== Making Agarose Gel ==
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[[Making Agar Plates]]
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* Add 20ml 50 X TAE to 980ml H2O.
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[[Isolating Plasmid from Cells (Miniprep)]]
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* Add 3g agarose powder to 300ml of the TAE-H2O (1 x TAE) solution.
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* Microwave full power for 5 minutes or until solution is clear and agarose has dissolved.
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* Leave to stand for 5-10 minutes. Put tape around sides of tray.
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*Pour solution into tray, add 4ul of ethidium bromide and mix gently using the comb.
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* Clean the comb (this prevents capillary action drawing the gel up between the comb teeth and making 'spikes' around the wells) and reinsert near one end of the tray.
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* Leave to set 30-40 mins.
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* Remove tape and comb, place tray in electrophoresis machine and pour 1 x TAE solution to just cover the gel.
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* Combine 1ul of loading buffer for every 5ul sample being loaded and mix by pipetting up and down.
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* Load marker and samples and run the gel.
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N.B. We have found that a thin gel works best for loading small samples, but a thicker gel may be preferable if lots of sample needs to be loaded (for example if a fragment needs to be cut from the gel).
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[[Restricting Plasmids (Double Restriction)]]
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N.B. We have run gels at 100V for 20mins and 70V for 60mins. The latter setting/time is better for running larger fragments such as whole plasmids as a high voltage can cause shearing of the DNA.
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[[Purifying DNA from an enzymatic reaction]]
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[[Cutting a Specific Band from Agarose Gel]]
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[[Purifying DNA from Gel Slices]]
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[[Ligating DNA]]
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[[Transforming into DH5α or TOP10 E. coli]]
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[[Making Frozen Glycerol Cell Stocks From Overnight Cultures]]
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[[Making Overnight Cultures from Frozen Glycerol Cell Stocks]]
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[[Making Overnight Cultures from Agar Colonies]]
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[[Transforming into Bacillus subtillis]]
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[[Inducing Bacillus subtilis with Subtilin]]
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[[Preparing Bacillus subtilis for Microscopy]]
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Latest revision as of 23:03, 30 September 2008

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