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| | {{:Team:Newcastle University/Header}} | | {{:Team:Newcastle University/Header}} |
| | {{:Team:Newcastle University/Template:UnderTheWetLab|page-title=[[Team:Newcastle University/Protocols|Protocols]]}} | | {{:Team:Newcastle University/Template:UnderTheWetLab|page-title=[[Team:Newcastle University/Protocols|Protocols]]}} |
| | + | {{:Team:Newcastle University/WetlabSideBar}} |
| | + | <div id="maincol"> |
| | + | ==Protocols== |
| | | | |
| | + | [[Agarose Gel Electrophoresis]] |
| | | | |
| - | == Agarose Gel Electrophoresis ==
| + | [[Making Agar Plates]] |
| | | | |
| - | * Add 20ml 50 X TAE to 980ml H2O.
| + | [[Isolating Plasmid from Cells (Miniprep)]] |
| - | * Add 3g agarose powder to 300ml of the TAE-H2O (1 x TAE) solution.
| + | |
| - | * Microwave full power for 5 minutes or until solution is clear and agarose has dissolved.
| + | |
| - | * Leave to stand for 5-10 minutes. Put tape around sides of tray.
| + | |
| - | * Pour solution into tray, add 4μl of ethidium bromide and mix gently using the comb.
| + | |
| - | * 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.
| + | |
| - | * Leave to set 30-40 mins.
| + | |
| - | * Remove tape and comb, place tray in electrophoresis machine and pour 1 x TAE solution to just cover the gel.
| + | |
| - | * Combine 1μl of loading buffer for every 5μl sample being loaded and mix by pipetting up and down.
| + | |
| - | * Load marker and samples and run the gel.
| + | |
| | | | |
| - | 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.
| + | [[Restricting Plasmids (Double Restriction)]] |
| | | | |
| - | 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.
| + | [[Purifying DNA from an enzymatic reaction]] |
| | | | |
| | + | [[Cutting a Specific Band from Agarose Gel]] |
| | | | |
| - | == Isolating Plasmid from Cells (Miniprep) ==
| + | [[Purifying DNA from Gel Slices]] |
| | | | |
| - | * Pellet overnight culture by centrifuging 13,000g for 10 minutes.
| + | [[Ligating DNA]] |
| - | * Pipette out supernatent and discard.
| + | |
| - | * Add 250μl resuspension buffer R3 and mix by pipetting up and down.
| + | |
| - | * Transfer to capped tubes.
| + | |
| - | * Add 250μl lysis buffer L7 and mix by inverting tube 5 times.
| + | |
| - | * Incubate for 5 minutes at room temperature.
| + | |
| - | * Add 350μl precipitation buffer. Invert until mixture is homogenous.
| + | |
| - | * Centrifuge 13,000g for 10 minutes. Place spin column into recovery tube.
| + | |
| - | * Load supernatent into spin column and discard capped tube.
| + | |
| - | * Centrifuge 13,000g for 1 minute. Discard supernatent.
| + | |
| - | * Add 700μl wash buffer W9 (with ethanol).
| + | |
| - | * Centrifuge 13,000g for 1 minute. Discard superantent.
| + | |
| - | * Centrifuge 13,000g for 1 minute to remove all liquid. Discard any remaining supernatent and recovery tube.
| + | |
| - | * Place spin column in new recovery tube.
| + | |
| - | * Add 100μl TE buffer or MilliQ H2O.
| + | |
| - | * Incubate for 1 minute at room temperature.
| + | |
| - | * Centrifuge 13,000g for 2 minutes. The supernatent in the recovery tube should contain isolated plasmid.
| + | |
| - | * Discard spin column and store plasmid at -20°C.
| + | |
| | | | |
| | + | [[Transforming into DH5α or TOP10 E. coli]] |
| | | | |
| - | == Restricting Plasmids (Double Restriction)==
| + | [[Making Frozen Glycerol Cell Stocks From Overnight Cultures]] |
| | | | |
| - | 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.
| + | [[Making Overnight Cultures from Frozen Glycerol Cell Stocks]] |
| | | | |
| - | * 46μl MillQ H2O
| + | [[Making Overnight Cultures from Agar Colonies]] |
| - | * 10μl 10 x buffer
| + | |
| - | * 40μl plasmid sample
| + | |
| - | * 2μl enzyme 1
| + | |
| - | * 2μl enzyme 2
| + | |
| - | Total volume = 100μl
| + | |
| | | | |
| - | '''Concentrated'''
| + | [[Transforming into Bacillus subtillis]] |
| - | * 8μl MilliQ H2O
| + | |
| - | * 5μl 10 X buffer
| + | |
| - | * 10μl plasmid sample
| + | |
| - | * 1μl enzyme 1
| + | |
| - | * 1μl enzyme 2
| + | |
| - | Total volume = 30μl
| + | |
| | | | |
| | + | [[Inducing Bacillus subtilis with Subtilin]] |
| | | | |
| - | * Incubate solutions for 90 minutes in a 37°C water bath.
| + | [[Preparing Bacillus subtilis for Microscopy]] |
| - | * 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.
| + | </div> |
| - | | + | |
| - | | + | |
| - | == Transforming Plasmid into Cells ==
| + | |
| - | | + | |
| - | * Add 2μl plasmid to 50μl of competent cells.
| + | |
| - | * Incubate for 30 minutes on ice.
| + | |
| - | * Heat shock for 2 minutes at 42°C.
| + | |
| - | * Add 250μl LB.
| + | |
| - | * Incubate for 120 minutes at 37°C whilst shaking or rotating.
| + | |
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