Melbourne/Preparing an agarose gel

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Latest revision as of 14:44, 28 September 2007

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• Applications:
  1. Diagnostic
  2. Gel purification

• Time to complete protocol:
  • Lab time: 15min,15min.
  • Waiting time:30min
• Approximate cost of materials: $

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls

• DNA grade Agarose
• 1x Gel Buffer (TAE Buffer or TBE depending on desire)
  • There is a stock of 10x TAE on the shelf above our bench
• Ethidium Bromide
  • Ethidium bromide is a carcinogen. Be sure to wear gloves when handling anything that may contain ethidium bromide. Wear gloves wherever handling the electrophoresis apparatus and throw them away when finished. Dispose of any waste that may contain ethidium bromide in the labelled ethidium bromide waste containers (eg. gel running buffer into the ethidium bromide liquid waste container)

Method including controls

For 0.8% gel

1. Calculate required amount of agarose and weigh out in small plastic tray on balance.
   • Large gel takes 100mL => 0.800g agarose
   • Small gel takes 50-60mL => 0.400-0.480g agarose
2. Put agarose in flask or shot bottle of sufficient volume to allow for boiling (at least 2x volume of buffer to be added)
3. Add necessary volume of 1x buffer (TAE is better if you are intending to gel purify and seems to give cleaner bands. Use measuring cylinder.
4. Disolve agarose by microwaving. This does not take long, watch to ensure that you don't make your agar boil over and make a mess. If this does happen clean it up immediately so that the agar doesn't set.
5. Ensure that your agar is competely dissolved.
6. Prepare mould for gel by either blocking ends with gel clamp or masking tape
7. Insert desired comb at one end of gel (approximately 1cm from gel end if there are no slots to designate where the comb goes)
8. Once the agar has cooled slightly, but before it sets add ethidium bromide (20uL/50mL gel) and swirl to mix.
9. Pour gel into mould smoothly, not too fast or you will get bubbles, not too slow or you will make a mess.
10. Balance a piece of paper over the top of the gel to prevent dust from settling on the gel.
11. Allow to set for approximately half an hour, you can check if its set by poking the courner with a pipette tip (but you really shouldn't)
12. If you used the gel vice to stopped the ends of the mould, carefully release the vice and move the mould to the electrophoresis tank with the comb/wells at the end with the black (negative electrode). If you used tape move on to the next step.
13. To remove the comb pour a small amount of 1xBuffer on the gel to wet the tops of the wells. Gently lift the comb from one side initially, once it has started to come up lift from both sides. Be careful not to break the gel or lift it away from the base.
    • If the comb is sticking to the gel use a small, clean spatula to encourage the gel away from the comb (similar to running a knife around the edges of a cake tip to help get it out but on a much smaller and less appertising scale). This should not happen if the comb is clean!!!
14. If you used tape to stopper the ends of the mould remove the tape over the electrophoresis tank so as any excess buffer is caught by the tank and you don't make a mess. Place the gel in the tank with the wells towards the negative/black electrode.
15. Fill the tank with the same 1x buffer as was used to prepare the gel, until the buffer covers the gel with a layer of 1-2mm.
16. Load the gel

Equipement Required

• Measuring cylinders
• Balance
• SmallPlastic measuring tray
• Glassware in which to dissove agarose
• Microwave
• Pipette and tips
• Gel mould and comb
• Masking tape or gel vice
• Electrophoresis tank

References