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.
- While allowing flask to cool, assemble gel apparatus by taping ends of tray (flush against bottom to prevent leakage) and inserting combs (20-well comb holds 12+ μL).
- Cool flask until it is hold-able.
- Add 50 μL EtBr, swirl to mix, and pour in tray & allow to solidify (15 min).
- Remove combs, put tray in electrophoresis apparatus, fill with appropriate buffer until gel is fully submerged
- Add 5 μL/ladder to gel (found in fridge H)
- Add 2 μL loading buffer to each sample and load on gel
- Run at 100 V (if switch is on low, read lower scale; don’t go too far over or gel will melt), until bands migrate approx. 2/3 down gel. (takes around 45 min)