AGAROSE (DNA) GEL PROTOCOL
- 1X TAE or 1X TBE
- Agarose (powder)
- Ethidium Bromide (EtBr)
- Make [x] % agarose gel (%= weight/volume) according to size DNA you want to separate (higher MW bands are better resolved by lower percentage gels, e.g. 0.6%, while lower MW bands are best resolved by higher percentage gels, e.g. 1.4%). For most applications, 1% gel is appropriate, so weigh 1g agarose, and place in a 250 mL erlenmeyer flask (or appropriately large glassware so that the solution won’t boil over).
- To it add 100 mL 1X TAE or 1X TBE (TAE is used when DNA will be extracted from gel, TBE is used for diagnostic purposes).
- Swirl to mix.
- Cover flask w/ Reynolds wrap & microwave for approx. 45 sec or until solution begins to boil.
- Cool flask until it is hold-able. Note: >55C may warp the tray.
- Pour into gel box and allow to solidify, don't forget the combs!
- Add x10 SYBR green to DNA and incubate for 15 in dark
- Add x5 loading buffer
- Add 2 μL loading buffer to each sample and load on gel
- Add samples to wells being care not to spill over into adjacent wells
- Run at 100 V until bands migrate approx. 2/3 down gel. (takes around 45 min)