268 Burrell
From the preceding discussion in this chapter, it is clearly important
that, to obtain reproducible results, the purity and concentration of the
nucleic acid used must be consistent, the supply and concentration of
RNase A standardized, the pH and the ionic strength standardized.
3.1. Materials for Mismatch Analysis- 32p-labeled RNA prepared for example with SP6 RNA polymerase (23).
Approximately 3 ng of probe RNA containing 1 x 10 -5 cpm is required
per hybridization. - RNase A at a concentration of 2 mg/mL in sterile distilled water and
boiled for 10 min (so that it is DNase free). - Deionized formamide.
- Hybridization buffer:
- 80% Formamide
- 40 mM PIPES (pH 6.4)
- 0.4 mM NaC1
- 1 mM EDTA
- RNase buffer:
- 10 mM Tris-HC1 (pH 7.5)
- 1 mM EDTA
- 200 mM NaCI
- 100mMLiCI
- 20% SDS.
- Proteinase K 10 mg/mL.
- Phenol:Chloroform (1:1) containing 4% isoamyl alcohol, 0.01%
hydroxyquinoline. - Carrier transfer RNA (e.g., yeast, wheat).
- 4M Ammonium acetate.
3.2. Methods for Mismatch Analysis- Suspend 20-50 ng of cloned test DNA or 3-6 ~tg of total genomic test
DNA in 30 laL of hybridization buffer. - Add 0.5 laL of probe RNA, mix the solutions, and centrifuge the drop-
lets to the bottom of the tube. - Heat to 90°C for 10 min.
- Anneal at 45°C for 30 min.
- Add 350 I.tL of RNase buffer containing RNase A at 40 ~tg/mL.
- Mix by vortexing and centrifuge droplets to bottom of tube.
- Incubate at 25°C for 30 min.
- Add 10 ~tL of 20% SDS and 10 ~tL of proteinase K (10 mg/mL).
- Mix and centrifuge droplets to bottom of the tube.