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(New page: ==Introduction== Agarose gels help you visualize DNA. Cool! You can make agarose from 0.5% to even 3%, by mass. 0.7% shows separation of large fragments (5-10kb) and 2% shows good separat...)
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Revision as of 15:35, 17 June 2008
Agarose gels help you visualize DNA. Cool!
You can make agarose from 0.5% to even 3%, by mass. 0.7% shows separation of large fragments (5-10kb) and 2% shows good separation of small fragments (0.2k-1k). Keep in mind the gelliness/solidness positively correlates with more agarose; 0.5% gel will be floppy and fragile.
- TAE or TBE, 1x - 50 mL is good for a little gel but you have to determine empirically
- Don't run a TAE gel in TBE
- Add extra volume of TAE to compensate for boiling
- If gel purifying: add 1 mmol/L guanosine to the TAE/TBE. This does not affect ligation and other downstream steps, however it will protect sticky ends. (See references)
- 0.5-2% agarose, by mass (e.g. for 1%, dissolve 0.5g agarose in 50 mL)
- Ethidium Bromide (for 10mg/mL, use 1uL per 50mL TAE)
- Pour TAE into a flask that you can swirl to mix the contents
- Add agarose and swirl vigorously. It will be cloudy
- Microwave until it boils. Take it out and swirl (caution it's hot). Keep microwaving until the agarose flakes are minimized.
- Let it cool somewhere until around 50 C - to minimize EtBr vapours
- Add EtBr and swirl
- Pour into a gel tray and let it solidify
- Load samples and run gel.
How fast will my gel run?
Higher voltage and your DNA will migrate faster. It will also heat up the buffer⋛ don't melt it!
(Please add your own results too!)
- 1% agarose 50mL with Qiagen GelPilot 5x - orange <100bp band hits bottom ~20 minutes at 150V