Team:Newcastle University/Protocols

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<div id="maincol">
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==Protocols==
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[[Agarose Gel Electrophoresis]]
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== Agarose Gel Electrophoresis ==
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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 4μl 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 1μl of loading buffer for every 5μl 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). A comb with teeth taped together using autoclave tape can also be used to create larger wells.
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[[Restricting Plasmids (Double Restriction)]]
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N.B. We have run gels at 100V for 20 minutes and 70V for 60 minutes. 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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== Isolating Plasmid from Cells (Miniprep) ==
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[[Purifying DNA from Gel Slices]]
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* Pellet overnight culture by centrifuging 13,000g for 10 minutes.
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[[Ligating DNA]]
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* Pipette out supernatent and discard.
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* Add 250μl resuspension buffer R3 and mix by pipetting up and down.
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* Transfer to capped tubes.
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* Add 250μl lysis buffer L7 and mix by inverting tube 5 times.
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* Incubate for 5 minutes at room temperature.
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* Add 350μl precipitation buffer. Invert until mixture is homogenous.
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* Centrifuge 13,000g for 10 minutes. Place spin column into recovery tube.
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* Load supernatent into spin column and discard capped tube.
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* Centrifuge 13,000g for 1 minute. Discard supernatent.
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* Add 700μl wash buffer W9 (with ethanol).
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* Centrifuge 13,000g for 1 minute. Discard superantent.
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* Centrifuge 13,000g for 1 minute to remove all liquid. Discard any remaining supernatent and recovery tube.
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* Place spin column in new recovery tube.
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* Add 100μl TE buffer or MilliQ H2O.
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* Incubate for 1 minute at room temperature.
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* Centrifuge 13,000g for 2 minutes. The supernatent in the recovery tube should contain isolated plasmid.
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* Discard spin column and store plasmid at -20°C.
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[[Transforming into DH5α or TOP10 E. coli]]
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== Restricting Plasmids (Double Restriction)==
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[[Making Frozen Glycerol Cell Stocks From Overnight Cultures]]
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We have conducted restrictions in varying concentrations and total volumes; however they all follow the same basic procedure. Below are two of the different restrictions we carried out.
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[[Making Overnight Cultures from Frozen Glycerol Cell Stocks]]
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* 46μl MillQ H2O
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[[Making Overnight Cultures from Agar Colonies]]
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* 10μl 10 x buffer
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* 40μl plasmid sample
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* 2μl enzyme 1
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* 2μl enzyme 2
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Total volume = 100μl
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'''Concentrated'''
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[[Transforming into Bacillus subtillis]]
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* 8μl MilliQ H2O
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* 5μl 10 X buffer
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* 10μl plasmid sample
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* 1μl enzyme 1
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* 1μl enzyme 2
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Total volume = 30μl
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[[Inducing Bacillus subtilis with Subtilin]]
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* Incubate solutions for 90 minutes in a  37°C water bath.
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[[Preparing Bacillus subtilis for Microscopy]]
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* If DNA is to be run on a gel and a band cut out, this can be done without purifying the DNA. For all other downstream applications or for storage, solution must be purified to remove the enzymes.
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</div>
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== Transforming Plasmid into Cells ==
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* Add 2μl plasmid to 50μl of competent cells.
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* Incubate for 30 minutes on ice.
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* Heat shock for 2 minutes at 42°C.
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* Add 250μl LB.
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* Incubate for 120 minutes at 37°C whilst shaking or rotating.
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Latest revision as of 23:03, 30 September 2008

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