- Formamide: good but not the highest grade (e.g. Sigma catalogue number F7508). Immediately after purchase, store at –20 °C in small aliquots for hybridization mixture preparation and larger aliquots sufficient for one washing, typically 40 ml (Note 1). Formamide is a potential carcinogen
- 20x SSC (Appendix 1): Filter (0.22 µm) sterilize if it has not been autoclaved
- 50% (v/v) dextran sulfate in water. Filter (0.22 µm) sterilize and store for up to a week at room temperature, longer at –20 °C (Note 2)
- 10% (w/v) SDS (sodium dodecyl sulfate, also called sodium lauryl sulfate) in water, filter sterilize. Store at room temperature
- Sonicated or autoclaved DNA from salmon or herring sperm or E. coli (1 µg/µl; treat DNA following Protocol 3.2, or purchase commercially). Fragments should be 100-300bp long. Store in aliquots at –20 °C
- Labeled and tested probe DNA (Chapter 4; typical experiments use 0.5 to 1 µl of resuspended probe per slide). Store at –20 °C
- Optionally, unlabeled autoclaved blocking DNA (Protocol 3.2). Fragments should be 100-300bp long. Store at –20 °C
- Calculate the content and quantity of hybridization mixture following Table 8.1 and record them in Form 8.1. Typical hybridization mixture volume is 30 µl per slide, but 20 – 40 µl can be used depending on area of preparation on slide
- For each probe or probe combination, prepare hybridization mixture in a microcentrifuge tube according to your calculations on Form 8.1 (Note 3).
- Mix well and denature by floating the sealed tube in a water bath at 70 ° C for 10 min. Place tubes on ice for 5-15 min .
- Proceed to Protocol 8.4 for denaturation and hybridization
- Molecular biology grade formamide will have a pH around 7 and be solid at –20 °C; it degrades at room temperature. Very high quality formamide can be purchased frozen, but for the protocols here, freezing immediately on receipt is adequate if fresh from the supplier. Formerly, an ion exchange resin (e.g. Amberlite, Merck) was added to purify crude grades not specified for molecular biology
- The solution is very difficult, but possible, to force through the filter. To pipette the dextran sulfate solution, cut off the end of the micropipette tip to give a larger orifice
- Nearly every slide will need a different mixture, depending on material and probe; however it is easier and often more accurate to make a ‘master mix' of components common to all hybridization mixtures and partition this before adding individual components
- The hybridization mixture containing formamide was first described by Harper et al . 1981. The protocol described here is based on Schwarzacher et al. (1992) and Heslop-Harrison et al. (1990).