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BNA experimental condition for FISH application

Can you suggest a BNA experimental condition for FISH application?

Example of experimental conditions for FISH

Information below is a different conditions one can try out with FISH technique using BNA/DNA mixmers as probes

Probe Amount:
  • 150 ng (20 pmoles)
  • 100 ng (13.4 pmoles)
  • 75 ng (10 pmoles)
  • 50 ng (6.4 pmoles)
Denaturation:
  • Separate denaturation of slide and probe at 75 °C for 5 min Simultaneous denaturation at 75 °C No denaturation
Hybridization mixture 
  • 50% formamide/2xSSC/10% dextran (pH 7.0)
  • 2xSSC/10% dextran sulfate (pH 7.0)
Hybridization time
  • Overnight
  • 5 h
  • 3 h
  • 1 h
  • 30 min
Hybridization temperature
  • Overnight
  • 5 h
  • 3 h
  • 1 h
  • 30 min
Post wash
  • Normal FISH wash
  • 60 °C 50% formamide
  • No formamide wash

The saline-sodium citrate (SSC) buffer is used as a hybridization buffer, to control stringency for washing steps in protocols for Southern blotting, in situ hybridization, DNA Microarray or Northern blotting. 20X SSC may be used to prevent drying of agarose gels during a vacuum transfer.
  • A 20X stock solution consists of 3 M sodium chloride and 300 mM trisodium citrate (adjusted to pH 7.0 with HCl).
  • A 2X SSC buffer consists of 0.3 M sodium chloride and 30.0 mM trisodium citrate (adjusted to pH 7.0 with HCl).
Procedure
 
FISH is carried out as described in Table 1. The amount of probe can be varied between 6.4, 10, 13.4 and 20 pmoles.
 
Denaturation of the target DNA and the probe is performed at 75 °C for 5 min either separately using 70% formamide or simultaneously under the coverslip in the presence of hybridization mix containing 50% formamide. In addition, effect of no denaturation is also tested. Two alternative hybridization mixtures can be used: 50% formamide/2xSSC (pH 7.0) / 10% dextran sulphate or 2xSSC (pH 7.0)/10% dextran sulphate.
 
Hybridization times include 30 min, 1, 2, 3 h and overnight. Hybridization temperatures include: 37, 55, 60 and 72 °C. Post washing is either done as for standard FISH, or with 50% formamide/2xSSC at 60 °C, or without formamide. Hybridization signals with biotin labeled BNA/DNA mixmers are visualized indirectly using two layers of fluorescein labeled avidin linked by a biotinylated anti-avidin molecule, which amplifies the signal 8–64 times. The hybridization of Cy3 labeled molecules, however, is visualized directly after a short washing procedure.
 
Slides can be mounted in Vectashield (http://www.vectorlabs.com/catalog.aspx?prodID=428) containing 40-60-diamidino-2-phenylindole (DAPI).
 
The whole procedure is carried out in the dark. The signals can be visualized using a Leica DMRB epifluorescence microscope equipped with a SenSys charge-coupled device camera (Photometrics, Tucson, AZ), and IPLAB Spectrum Quips FISH software (Applied Imaging international Ltd, Newcastle, UK) within 2 days after hybridization.
 
In general, twenty metaphases are analyzed after each hybridization experiment.

References:
  1. Silahtaroglu AN, Hacihanefioglu S, Guven GS, Cenani A, Wirth J, Tommerup N, Tumer Z. Not para-, not peri-, but centric inversion of chromosome 12. J Med Genet 1998;35(8):682–4.