HELP needed for Telomere Length Chemiluminescence Assay

[justskipee]

Hello there,

 

We are using the Roche TeloTAGGG Telomere Length Assay to measure telomere lenth in peripheral blood mononucleocytes. (manual is found at https://www.roche-applied-science.com/pack-insert/2209136a.pdf).

 

It is a non-radioactive chemiluminescent assay, that involves digestions, gel electrophoresis, telomere probing...

 

We have been having a very weird problem. Our film shows nice smears for each lane, but it is like a small river flowed across the lanes. No one has a clue what would cause it, and it has been very frusterating given the cost and time we have used already.

 

Here is a link to a picture of our film.

 

Does anyone have any clue what could be causing this problem????

 

Thanks so much for your help,

Justin

[MedGen]

I'm not familiar with chemiluminescence visualisation, but that looks like it might be a problem with the camera rather than the gel or the assay. I say that because it looks like it is overlaid on the gel, you can still see through it where the band smears are. What system are you using to visualise the gel with?

 

i.e. BioRad Geldoc, etc.

[CharonY]

Actually it looks a bit like something was on the film, or the developer was not stopped evenly, something like that. Is this reproducible?

[justskipee]

Charon - We did about 4 films and they all had the same pattern, so it is reproducible. When we slightly overexposed it (example posted) we could see the lanes underneath. We have had successful attempts 4 months ago, so this has caught us off guard.

 

MedGen - We placed the membrane with fuji film medical x-ray film in an autoradiography cassette.

 

 

Here is a summary of the steps involved. Full instructions:

http://www.roche-applied-science.com/pack-insert/2209136a.pdf

 

When we place the membrane into the opened hybridization bag, we then put 40 drops of sustrate solution. We didn't see any excess pooling or anything that would correspond to the "river" looking pattern.

 

Do the assay instructions above help at all? Thanks for your help!

[CharonY]

Reproducible. In that case is it possible that the films are no good anymore? You could try and expose a film and develop it to see whether you see a similar pattern on it.