Hi Saeede,

 we used this protocol with great results. See Ladetto M et al Blood 2004.

TRF length was determined using Southern blot analysis according to the following procedure. Aliquots of 2 μg DNA were digested by mixing HinfI (20 MU) and RsaI (20 MU) (Roche Diagnostics, Mannheim, Germany) for 2 hours at 37°C, according to the manufacturer's recommendations. TRF were then separated by 0.8% agarose gel electrophoresis in 1 × TAE buffer (pH 8.0). Gels were transferred to a positively charged nylon membrane (catalog no. 1 417 240; Roche Diagnostics) and were ultraviolet (UV) light cross-linked for 10 minutes. Hybridization was performed with the TeloTAGG Telomere Length Assay Kit (Roche Diagnostics), which contains all the reagents required. Membranes were prehybridized for 2 hours in the prehybridization solution at 62°C and then hybridized for 3 hours in the same conditions by adding 2 μL of the digoxigenin (DIG)-labeled telomere-specific probe. After hybridization, filters were washed twice at room temperature for 15 minutes in 2 × washing solution and then twice at 39C° in 0.5 × washing solution for 20 minutes. Filters were then incubated with a DIG-specific antibody covalently coupled to alkaline phosphatase (AP). Finally, results were visualized using AP metabolizing CDP-Star, a highly sensitive chemiluminescent substrate. The light signal produced on the site of the hybridized probe was recorded on x-ray film (Lumi-Film Chemiluminescent Detection Film; Roche Diagnostics). Each assay was performed twice. Membranes were then scanned and analyzed with Kodak Digital Science 1D Image Analysis Software (Scientific Imaging Systems, New Haven, CT).

Tricky points are: amount of input DNA, incubation temp during hybridization and Ph off all solutions

Good luck!

Daniela

I agree with Daniela's comments. We also used the kit and followed the protocol that was provided. The quality of the DNA, the extent of UV-cross-linking to the membrane and the extent of hybridization all contribute to the final outcome. Therefore, practically speaking, performing a trial run with positive control should work before using unknown or test samples.

As both Daniela and Divaker have said, I have used the kit to great effect as well.  Have you tried increasing the time of transfer to the positive nylon?

As to the cost of the kit, all of the components can be purchased from various companies (Roche sells a nice set of wash and block buffers, we use IDT for the probe and NEB for the enzymes.  Obviously other companies also sell, and you can make a lot of the buffers from scratch as well) that cuts the cost of the protocol significantly.