Project:Gel electrophoresis box

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

13 April 2011: Photos of the first Electrophoresis experiment

People involved

Bugs
Martind

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