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Team:Newcastle University/Protocols
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* Microwave full power for 5 minutes or until solution is clear and agarose has dissolved. | * 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. | * 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. | + | * 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. | * 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. | * Leave to set 30-40 mins. | ||
| Line 28: | Line 28: | ||
* Transfer to capped tubes. | * Transfer to capped tubes. | ||
* Add 250μl lysis buffer L7 and mix by inverting tube 5 times. | * 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. | * Add 350μl precipitation buffer. Invert until mixture is homogenous. | ||
| - | * Centrifuge 13,000g for 10 minutes. | + | * 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 -20oC. | ||
Revision as of 11:09, 27 August 2008
Newcastle University
GOLD MEDAL WINNER 2008
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Home >> Protocols
Agarose Gel Electrophoresis
- Add 20ml 50 X TAE to 980ml H2O.
- 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).
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.
Isolating Plasmid from Cells (Miniprep)
- Pellet overnight culture by centrifuging 13,000g for 10 minutes.
- 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 -20oC.
