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During this interim period donations and continued membership are greatly appreciated while we transition to our new space.
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
- 13 April 2011: Photos of the first Electrophoresis experiment
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)
- 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
- 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!