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Hybridization buffer
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Two types of hybridization buffer, one regular and one concentrated, were prepared in our laboratory and were used to resuspend all labeled DNA probes (Table 6). Aliquots of these buffers could be stored at -20° C for years.
Quality of the hybridization buffer
New hybridization buffers prepared in the laboratory need needs to be tested for their performance. We usually prepare three or four, 10-20 ml each, hybridization buffers at one time, using different formamide sources. The buffer is aliquoted and stored at -20° C. Each batches is tested and compared with a "good" buffer(s), still in use in the lab. Results of such experiments are shown in Fig. 7e-g. When slides were prepared, pretreated and hybridized with the same probe using identical conditions and varying only the hybridization buffer(s), differences were apparent. Although hybridization of a cosmid probe worked in all three cases, signal to noise ratio was much better in Fig.7g than in the first two pictures. A less then optimal cell suspension, with more cytoplasmic debris is recommended for such a test, as it is more difficult to hybridize onto and will reveal easier any problems with the hybridization buffer. The main parameter influencing the quality of the buffer seemed to be the formamide freshness and purity. A high quality formamide taken from a new bottle is recommended for buffer preparation.
FISH with small amounts of labeled probe
The regular hybrodization buffer was used for probes requiring small amounts of labeled DNA (plasmids, cosmids, BACs). For such situations, a solution of concentrated competitor (Cot1) DNA, 10-20µg/µl in hybridization buffer, was also previously prepared. 1-3 µl labeled probe (nick translation or PCR) were mixed with 1-2 µl concentrated Cot1 DNA and with 8-10 µl regular hybridization buffer, to bring the final volume to about 12-13 µl. This volume was used for a 22x22 mm hybridization area.
FISH with large amounts of labeled probe.
For FISH procedures requiring large amounts of labeled DNA probe (YACs, CGH, M-FISH), precipitation and concentration of the labeled DNA was obligatory. In those cases, probes were mixed together with the appropriate amount of competitor DNA and ethanol precipitated. Vials were kept 5-10 minutes at room temperature, and were centrifuged 8-10 minutes at 14,000 rpm in a microfuge. When the pellet was large (included residual salt, EDTA, proteins), it was resuspended in 100 µl water. Ethanol-precipitated was performed again, after adding 10 ul 3M NaAcetate pH5.2 to the DNA. The DNA pellet can be dried 5-10 minutes on a metal block at 50-60° C, and resuspended in 4-4.5 µl 1xSSC at 50-60° C. The resuspended DNA is mixed with 7-8 µl concentrated hybridization buffer using a pipette. The DNA can be denatured and used for hybridizations. This procedure allowed large DNA pellets to dissolve easily, and was useful especially in with MFISH probes.