Melbourne/Loading a DNA gel

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< Melbourne(Difference between revisions)
 
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=====Primary & secondary Reagents Required including controls=====
=====Primary & secondary Reagents Required including controls=====
*[[Melbourne/Preparing an agarose gel|0.8% agarose gel]]
*[[Melbourne/Preparing an agarose gel|0.8% agarose gel]]
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*1x buffer (same as used to prepare the gel, [[Melbourne/Secondary Reagent TAE|TAE]]gives cleaner bands and is better for gel purification than TBE)
+
*1x buffer (same as used to prepare the gel, [[Melbourne/Secondary Reagent TAE|TAE]] gives cleaner bands and is better for gel purification than TBE)
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*[[Melbourne/primary DNA marker|DNA ladder]] with *[[Melbourne/primary dna loading|dye]]added (stock of 1kb ladder diluted 1 in 21 in loading dye is in the -20 freezer in iGEM box 1)
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*[[Melbourne/primary DNA marker|DNA ladder]] with [[Melbourne/primary dna loading|dye]] added (stock of 1kb ladder diluted 1 in 21 in loading dye is in the -20 freezer in iGEM box 1)
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*DNA to be run with loading *[[Melbourne/primary dna loading|dye]]already added.
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*DNA to be run with loading [[Melbourne/primary dna loading|dye]] already added.
=====Method including controls=====
=====Method including controls=====

Latest revision as of 15:02, 28 September 2007

<Return to list of protocols> <Team home page>

  • Applications:
    1. Diagnostic
    2. Gel Purification
  • Time to complete protocol:
    • Lab time: 15min.
    • Waiting time:30min-2hrs
  • Approximate cost of materials: $

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls
  • 0.8% agarose gel
  • 1x buffer (same as used to prepare the gel, TAE gives cleaner bands and is better for gel purification than TBE)
  • DNA ladder with dye added (stock of 1kb ladder diluted 1 in 21 in loading dye is in the -20 freezer in iGEM box 1)
  • DNA to be run with loading dye already added.
Method including controls
  1. Prepare gel and place in electrophoresis tank with the wells towards the black/negative electrode.
  2. Cover with 1x buffer such that there is a 1-2mm layer of buffer covering the gel and the wells are full.
  3. In lane one load 20uL of the ladder DNA
  4. The amount of DNA loaded varies depending on concentration, for the diagnostic digests load 20uL.
    • Ensure that you write down the loading order.
  5. If you have sufficient lanes load another 20uL of the ladder in the final lane for greater ease in interpretation.
  6. Place the cover on the electrophoeresis tank. Make sure that you connect black to black and red to red and plug the cables into the power supply.
  7. Set the voltage on the power supply and start. Check the gel after a few minutes to make sure it is running in the correct direction.
  8. Leave the gel running for about an hour then stop the power source or unplug your tank if another tank is using the same source and remove the gel from the bath(still in mould) Check the resolution of the bands by placing on UV light source next to microwave.
  9. If there is not enough resolution continue running the gel until suffiecient is reached. Be careful not to forget the gel and run your DNA off the end of the gel.
  10. If there is sufficient resolution take the gel(in the mould, in a container) to the gel photographing machine in the Cheng lab and photograph the gel.
  11. Dispose of the gel in the ethidium bromide waste bin and the buffer in the liquid ethidium bromide waste.

Ensure that you clean all apparatus that you use

Equipement Required
  • Pipette and tips
  • Electrophoresis tank
  • Power source
  • Transport container
  • UV light source
  • Gel Photographing machine
References


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