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Project:Gel electrophoresis box

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We made our own electrophoresis box with a laser cutter. All sides are 3mm perspex

Measurements for the outer box:

   Outer length: 167mm
   Inner length: 161mm
   Outer width: 107mm
   Inner width: 101mm
   Height including legs: 70mm
   Outer height of box: 41mm
   Inner height of box: 37mm
   Inner height to top holes (for electrodes): 20mm

Measurements for the inner box:

   Length: 99mm
   Outer width: 100mm
   Inner width: 94mm
   Outer height: 23mm
   Inner height: 20mm

Testing

People involved

Setup

Reagents: 10x TBE (NBSBio) Agarose (NBSBio) SafeWhite loading dye/DNA stain (NBSBio) 2kb ladder (NBSBio) Tapwater cut plasmid DNA from Bugs' lab

Attempting to make 1% agarose solution (i.e. 1g agarose in 100ml TBE buffer). Weighed 1g agrose into container. Mixed with 10ml of 10xTBE (measured by syringe) + 90ml tap water (measured by weight?), melted in microwave. By eye, judged to be way too thick; scale is inaccurate for such small weights? Added a further 10ml 10xTBE + 90ml water to dilute the agarose solution to something closer to 1% w/v. Microwaved again to ensure even mixing.

Used brown parcel tape to seal open ends of casting tray & put comb in position. Poured approx 100ml agarose into tray and allowed to set in 'fridge. NB: 100ml was probably too much, gave us a thick gel.

Filled gel running tank with 1x TBE (NB: what volume did we use?). Removed parcel tape from ends and immersed set gel in buffer.

Prepared samples: 10ul of DNA soluion + 2ul of loading dye, mixed by tapping. Samples loaded into wells.

Gels run at approx 120V [NB: What was the powerpack setup?] [What was the current?]

Tried to visualise DNA using UV LED from maplin (peak emission 450nm) Also tried to visualise using UV PCB exposure box (emission unknown)

Results

  • Sample loading worked
  • Loading dye ran with the current as expected.
  • Positive electrode reacted with something in buffer; lots of (very pretty!) blue crystals formed, electrode eaten away. Didn't seem to affect gel running speed, but we should probably try other electrode materials.
  • Couldn't see any stained DNA using UV LED (in darkish room)
  • Couldn't see any stained DNA using UV lightbox (in darkish room)
  • Bugs took the gel to his lab to visualise. It worked! Poor quality gel photo [here], better quality uploaded when lab scanner gets fixed.
  • Visible band is a piece of DNA that Bugs happens to know is approx 7kb (=7000bp = 7000 bases = 7000 nucleotides) long.
  • Ladder is very faint, but just about visible.
  • Looking at the photo above, I (Bugs) am pretty sure that the gel was more than 1% agarose. Not a problem in itself, but means that large pieces of DNA

Next Steps

  • Need better measurement of how much agarose we're putting in. Try doing it by volume e.g. 0.5 teaspoon / 100ml? Bugs will try to come up with a useful metric using equipment in work.
  • Use more ladder to make it brighter on picture
  • Need to find a better UV source to visualise results. Ideally approx 300nm.
  • Alternatively, explore DNA stains that don't need UV to visualise.
  • Try different materials for electrodes. Professional boxes use platinum, but there must be something a bit cheaper that's good enough!


Ideas for improvements

Open Source Electrophoresis tanks - They sell kits, but we can probably just use their published plans to laser-cut our own.
Instructions to build your own.

DIYBio thread on building gel box