Basic Ingredients and Instrumentation

1. Slides containing ethanol-fixed cells (or standard chromosome spreads).

2. 37°C humidified incubator.

3. Cover slips of appropriate size (e.g., 24 x 60 mm to completely cover a standard slide).

4. Coplin jars or other suitable containers for washing the slides.

5. 10X phosphate-buffered saline (PBS) and/or 20X standard saline citrate (SSC; PBS and SSC may to some extent replace each other in the buffers used).

6. Restriction enzyme and (10X) buffer (e.g., Roche).

7. Bovine serum albumin (BSA), DNase free.

9. Formamide (fresh or deionized).

10. Padlock probe(s).

11. T4 DNA ligase and 10X buffer (enzyme and buffer are supplied together by Roche).

Padlock Rolling Circle
Fig. 1. Rolling-circle PRINS from padlock probes. (Please see color insert following p. 48.)

12. 9-29 DNA polymerase and 10X buffer (enzyme and buffer are supplied together by Fermentas).

13. dATP, dCTP, dGTP dTTP: 100 mM(Lithium salts) from Roche.

15. Identifier oligonucleotides.

16. Fluorochrome labeled anti-digoxigenin for detection of digoxigenin-labeled DNA (Fab fragment, Roche). Fluorochrome-labeled Streptavidin for detection of biotin-labeled DNA (Roche, Vector Laboratories); optional.

17. Antifade mounting medium (Vectashield from Vector Laboratories or p-phe-nylenediamine from Sigma) containing either of the following:

18. 0.4 |M DAPI (Sigma; for blue counterstaining of DNA) or

19. 0.5 |g/mL Propidium iodide (Sigma) for red counterstaining of DNA.

20. Fluorescence microscope with standard excitation and emission filters (e.g., 81000 filter set from Chroma Technology).

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