Nick translation/PCR labeling
Note: a somewhat more detailed description of the nick translation and DOP-PCR labeling protocol, using custom-prepared or commercial fluor-dUTPs, is presented here. (Some of the data is redundant as it was posted at different times.)
Numerous DNA probes can be labeled by either nick translation or PCR (Table 4). The relative amount of each of these DNA probes (and the corresponding amount of Cot1 DNA) for one FISH experiment is detailed in Table 5.
In nick translation (Fig. 5a), which usually takes place at 15° C for 2 hours, final DNA size is controlled by carefully choosing the DNase concentration in the labeling reaction.
Primers used for DOP-PCR:
6MW = 5' ccg act cga gnn nnn nat gtg g 3' (primer initially designed in 1992 by A. Telenius)
Primer A is used for a few low-annealing temperature cycles (15-30 C), then primer B is added (binds to A) and cycles performed at higher annealing temperatures (55 C)
A = 5' tgg tag ctc ttg atc ann nnn n 3' (primers initially designed by S. Bohlander)
Tests to check labeling efficiency
The FISH assay.
For this assay, a small amount of a repetitive probe is labeled using the same reagents used for labeling the rest of the probes.A small amount of the repetitive probe is mixed in 10-11 ?l hybridization buffer, placed on a regular slide, and simultaneously denatured. The probe is allowed to hybridize for 20-30 minutes, after which the slide is rinsed in the usual solutions but for very short time (1-2 minutes each wash). If the probe was labeled with fluorescent nucleotides, it can be directly visualized at the microscope, otherwise a layer of the appropriate fluorescent antibody is added first. This testing procedure is faster than the dot blot assay and is very reliable.
The dot blot assay.
For the dot blot assay a small sample of labeled DNA is serially diluted to 1ng/?l, 100 pg/?l, 10 pg/?l and 1pg/?l. One ?l of each such dilution is spotted with a pipette tip on a small piece of nitrocellulose or nylon membrane, which is baked 30 minutes at 80° C. The membrane is soaked 1-2 minutes in a solution of 100 mM Tris (pH 7.5), 100 mM NaCl and 0.05% Triton-X-100 and then incubated for 15-30 minutes in blocking buffer (3% BSA in the same solution). The membrane is then incubated in a solution containing the appropriate dilution (usually 1:100 to 1:500) of an alkaline-phosphatase labeled antibody, against the fluorophore or haptene (BIO, DIG, DNP) previously used to label the DNA. After 5-10 minutes incubation, the membrane is rinsed in a buffer with a high pH (100 mM Tris, pH 9.7, 100 mM NaCl) and then incubated in the dark in a solution of NBT/BCIP as recommended by the vendor (Sigma). After 30-60 minutes the membrane is rinsed in TE buffer and dried. If the labeling is good, even 1pg labeled DNA should be visible (