Team:Newcastle University/Protocols

From 2008.igem.org

(Difference between revisions)
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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.
-
* Leave to incubate for 5 minutes at room temperature.
+
* 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

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Newcastle University

GOLD MEDAL WINNER 2008

Home Team Original Aims Software Modelling Proof of Concept Brick Wet Lab Conclusions


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.