DNA Spreading and Shodowing

DNA SPREADING AND SHADOWING

Grids

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