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Buffer Used In BNA Experiments

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07/01/2013

Buffer Used In BNA Experiments

Buffers used in BNA experiments

Application Concentration Buffer Reference
Antisense BNAs 1 micomolar (1 µM) 20 mM sodium phosphate, 100 mM NaCl pH 7.2 Obika et al., 2001
Duplex formation Duplex concentration 4 micromolar (µM) 100 mM NaCl, 10 mM sodium phosphate pH 7.2 Obika et al., 1998
Duplex formation 4 microM (µM) per strand 100 mM NaCl, 10 mM sodium phosphate pH 7.2 Imanishi and Obika 2002
Duplex formation 4 micromolar (µM) 100 mM NaCl, 10 mM sodium phosphate pH 7.2 Miyashita et al., 2007
Triplex formation > 0.2 µM

140 mM KCl, 10 mM MgCl2, 7 mM sodium phosphate pH 7.0

50 mM Tris-acetate, 100 mM MgCl2 pH 7.0

10 mM sodium cacodylate/cacodylic acid pH 6.8 200 mM NaCl, 20 mM MgCl2

Hari et al., 2003


Torigoe et al., 2011
RNAi

1 µM

Transfection:

0.2 to 20 nM

100 mM NaCl, 10 mM sodium phosphate pH 7.2

Transfection: Lipofectamine 2000 in OPTI-MEM buffer

Rahman et al. 2010,

Yamamoto et al. 2012

Example of experimental conditions for FISH

Table 1: Different conditions can be tried 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 XoC for 5 min

Simultaneous denaturation at 75 oC

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

RT

37 oC

55 oC

60 oC

72 oC
Post wash

Normal FISH wash

60 oC 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 oC 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 oC. Post washing is either done as for standard FISH, or with 50% formamide/2xSSC at 60 oC, 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 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.

Reference

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.