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ANITA RAUCH
- 20xSSC: 87.65 g NaCl plus 44.1 g Na-citrate plus 500 ml aqua bidest,
adjust pH to 7.0 with HCI 32%, store at room temperature
- PBS: 50 ml10xPBS plus 450 ml aqua bidest
- * 10 x PBS: 80 g NaCl plus 2 g KCl plus 11.5 g Na2HP04 plus 2 g KH 2 P0 4
add 11 aqua bidest, adjust pH to 7.0 with NaOH, store at room temperature
- 1 M MgCh: dissolve 20.3 g MgCh in 100 ml aqua bidest, store at room
temperature
- PBS-MgCh: 10 ml 1M MgCh plus 190 ml PBS
- 1% Formaldehyde: 2.7 ml formaldehyde (37%, Merck) plus 97.3 ml
PBS-MgC1 2
Denaturation - 70% formamide/2 x SSC: x per slide: 70 J..lI deionized formamide plus
10 J..ll phosphate buffer plus 10 J..l120xSSC plus 10 J..lI aqua bidest (may
be stored at -20
D e)
- Phosphate buffer: dissolve 0.7 g sodium dihydrogen phosphate monohydrate in 10 ml aqua bidest, dissolve 0.89 g sodium dihydrogen phosphate dihydrate in 10 ml aqua bidest and mix both, store at -20
D C in
portions of 1 ml.
* Deionized formamide: add 2.5 g ion exchanger (mixed bed resin) to
50 ml formamide, stir for 2 h, let the ion exchanger settle down and
aliquot formamide in 1 ml portions, store at -20
D C.
Hybridization mix - Commercially available direct labeled probes are used with the hybridization mix provided, following supplier's instructions.
- Self-labeled probes (e.g. with Roche Corp. Nick Translation Mix according to supplier's instructions) use with 20% dextran sulfate: dissolve 2 g dextran sulphate in 5 ml deionized formamide plus 1 ml phosphate buffer plus 1 ml 20xSSC plus 3 ml aqua bidest, store at -20
D
e.
Washing solutions - lxSSC: add 25 m120xSSC to 475 ml aqua bidest, adjust pH to 7.0-7.5
with 5 drops of 1 M HCl. Store at room temperature.
- 2xSSC/0.l% NP-40: add 50 ml 20xSSC to 450 ml aqua bidest, add
0.5 ml NP-40, adjust pH to 7.0-7.5 with 1 drop of HCI 32%