DNA Spreading and Shadowing
- Use ultrapure H2O for all solutions.
- Fill clean plastic or acid-washed petri dish with 0.15 M NH4Ac (hypophase) until surface is convex. Acid clean microscope slide at least 1 min. Rinse thoroughly in tap water followed by dH2O.
- Immerse slide in hypophase holding with acid-cleaned forceps.
- Sprinkle surface of hypophase with talc.
- Wipe 2 clean teflon bar with several fresh dry kimwipes.
- Slide bars across hypophase to push talc and dirt to one side.
- Mix hyperphase on piece of parafilm:
- DNA to give final conc of about 3 mg/ml
- cytochrome c: 2 ml
- NH4Ac: 50 ml
- ultrapure H2O to give final vol of 100 ml
- Slide second teflon bar away from first teflon bar.
- Pull slide out of hypophase with acid-cleaned forceps and prop on side of petri dish.
- Slide second teflon bar toward slide until it is separated from slide by distance of about 1 in.
- Sprinkle small amount of talc or graphite in space between 2 teflon bars using shaker or camel hair brush.
- Pipet 50 ml onto wet slide near interface evenly and slowly over 30-40 sec.
- With coated side down, bend edge of coated grid.
- Touch coated grid to powder-free area 60-90 sec after spreading DNA.
- Immediately dip grid into uranyl acetate for 20 sec.
- Wash 3-5 sec in 90% ethanol.
- Dry grid by touching edge of grid to kimwipe or bibulous paper.
- Pick up additional samples at points at least 1 cm apart.
- Set angle of tungsten wire with platinum/palladium at angle of 7°.
- Rotate stage during metal evaporation.
- Do not coat with carbon.