Prepare a 2% agarose gel (125 ml slab)
- Weight 2.5 agarose into beaker
- Fill up to 125 g with 0.5x TBE-Puffer
- Boil in microwave until agarose is dissolved (2 min)
- Some water may evaporate through heat. Add ddH2O until back up to 125 grams (apply water to sides of hot beaker)
- Cool till hand warm
- Add 1 ml of 1.375 MgCl2 solution
- Add 7 μL ethidium bromid from 10 mg/ml stock solution. (use gloves for this protocol. minimize exposure)
- Fill gel tray and install desired comb.
- ONce solid, fill gel box with TBE/11 mM MgCL2 buffer.
- Remove comb.
- Sample: mix 12 μL of each DNA sample with 3 μL 6x loading dye. Load into lanes
- Unfolded Plasmid: mix 1.2 μL of phage DNA (100 nM) with 10.8 μL ddH2O and 3 μL 6x-loading dye. Load into lanes
- DNA ladder: add 6 μL of 1kb DNA ladder (New England Biolabs)
- Put gel box into ice water bath
- Apply constant 70 V across the gel.
- Run for 3-4 hours.
- Can image using UV illuminator in dark room. Get picture>exposure>take picture>save under C: share files.
- Use UV transilluminator for band visulaization, and cut out the desired band with razor bands.
- Put slice of gel into tube.
- Spin down the debris.
- Cut off debris containing itp of the tub.
Invert tip into a freeze'n'squeeze spin column (Biorad).
- Spin 12 min at 12,000G in tabletop centrifuge.
- Throw away the top portion of the squeeze spin column.
- Label and store purified DNA at 4°C