Specimen Preparation

DNA Spreading and Shadowing

Grids

  • Acid clean grids.
  • Coat grids with Parlodion.

Solutions

  • Cytochrome c: 5 mg/ml in ultrapure H2O
  • NH4Ac: 2M in ultrapure H2O
  • 90% Ethanol: dilute 100% ethanol in glass bottle with ultrapure H2O
  • Uranyl acetate: 50 mM uranyl acetate in 50 mM HCl
    • Mix 10 ml uranyl acetate in 5 ml 90% ethanol

Spreading

  • 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.

Shadowing

  • Set angle of tungsten wire with platinum/palladium at angle of 7°.
  • Rotate stage during metal evaporation.
  • Do not coat with carbon.