DNA Electrophoresis

1. Make up agarose solution; need ~100ml for small gel or ~200ml for large gel. Percentage agarose needs to be appropriate for sizes of DNA fragments to be separated. Weigh agarose into weigh boat on balance. Pour into Duran bottle. Add appropriate volume of ½ x TBE buffer NOTE: After taking buffer, remember to turn bottle so tap is back over drip tray. NOTE: The higher the percentage of agarose, the less separation of larger fragments. e.g. separating ~0.9kb and ~5kb therefore making an 0.8% agarose gel = 0.8 mg agarose in 100 ml ½ xTBE buffer.
   
   
2. Tighten lid on Duran bottle and then release by ½ turn. Place in microwave on high for 2 minutes. Carefully tighten lid and remove from microwave by lid – CAUTION! VERY HOT! Ensure all the agarose has dissolved by swirling the bottle. NOTE: Make sure lid is not so loose it will be pushed off as solution boils, but not too tight that pressure can’t be released.
   
   
3. Allow the solution to cool to 50-60⁰c by placing in 55⁰c water bath for ~30 mins. NOTE: Alternatively, if using immediately, allow to cool slightly on bench before running under cold tap. Ensure the solution is moving inside the bottle to prevent localised setting of the gel in the bottle.
   
   
4. Tightly seal the ends of the gel tray either using rubber tubes or autoclave tape. Insert a comb with appropriate number of teeth for the desired number of wells.
   
   
5. Pour agarose gel into gel tray. Allow to set on the bench for ~15mins (small gel) or ~30mins (large gel). NOTE: Ensure gel is close to, but not higher than the top of the comb indentations.
   
   
6. When set, remove seal and place gel tray in DNA gel tank. Gently remove comb. Add enough ½ x TBE buffer to tank to just cover gel and fill all the wells. NOTE: Do not remove tape before carrying the gel tray over to the electrophoresis bench – the gel is slippery and may slide out of tray if tipped. NOTE: TBE buffer crystallises when is dries. Rinse everything that has had TBE buffer on it before it dries.
   
   
7. Add 5 μl of loading dye (New England BioLabs) to each DNA sample. Bench centrifuge for a few seconds to mix thoroughly. If samples are in PCR tubes place whole PCR tube in lidless Eppendorfs in order to centrifuge. NOTE: Double check you are not accidentally adding the DNA ladder.
   
   
8. Load 10 μl of DNA ladder (New England BioLabs) into first well. Gently load one DNA sample per well. Make a note of the contents of each well. NOTE: Wells can take up to 30μl of sample, 15μl should be sufficient for extraction. Consider retaining some of sample in case mistakes are made alter in extraction procedure. NOTE: Load fragments of different expected sizes in neighbouring wells to make cutting bands out of the well for easier.
   
   
9. Place lid on gel tank and connect leads up to the 500V power pack. Ensure this is connected up the right way round so that the DNA will move in the right direction through the gel. Turn on power pack at wall and on unit. Press ‘Set’ and adjust voltage using dial to 100V, press ‘Set’ again. NOTE: If then ‘Leakage’ light is on, there is too much buffer in the tank and it is short-circuiting the contacts. Turn off power pack, remove some buffer and re-start.
   
   
10. After a 10 minutes check that dye is running in correct direction. Leave to run for 1 hour (small gel).
    
    
11. Turn off power pack on unit and on wall. Remove tank lid.