Hi Valerie,

First try to blast your GFP probe sequences against the genome sequences (human?). you probe might contain sequences homologous to the wt genome;

Second, use repeatmasker (http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker) to analyze you probe with the related genome. The most likely cause for this smear is that you might contain repeated elements in you probe that detect numerous place at the genome including the marker;

Last, try to optimize you washing temperature.

Good luck with your SB.

KR,

Alun

Hi Valerie,

first of all your DNA seem not to be digested very good. I took your image as a copy of your agarose gel, and most of ypour DNA is still near the comb. I don`t know what kind of band you expect or what you are looking for, but if you want detect only a few amounts of DNA you will run into trouble.

I would also change the settings for pre-hybridisation to saturate unspecific binding and/or do the hybridisation at higher temperature.

I`m versed in southern blots with radioactive labeled probes, but I think that the basic things are the same also for diferent labeling procedures.

Good luck

Sibylle

Alun, thank you for the repeatmaster site. I did the blast and there was no repetitive sequence detected with the mouse genome.

Sibylle,

the first gel I posted is of the southern blot. I have attached an image of the agarose gel. It does seem like the probe detects the higher molecular weight fragments. I tried repeating the stringency washes on this blot yesterday - 60C for 1 hour shaking, but it didn't seem to decrease the noise.

maybe I should switch to a radioactive label? I've never used one and our lab is not "cleared" for radioactivity, but I'm starting to think that might be the best option. Do you use a kit to label your probe?

thanks again for all the help!

val

Please note that the southern blot image that i posted with the question is upside down. :)

What's the quality of your probe like when you run it on a gel? Is it a nice clean single PCR product? How did you purify it after the PCR?

here's a segment of my notebook for the trouble shooting. which may be helpful.

Peter, the band is clean and specific (there's an example image in my attachment). To prep the probe I run the PCR product out in a gel, excise it, and prep it with a Qiagen gel extraction kit. I've also used WT gDNA as a template to see if there would be any non specific PCR products, but there were none.

thanks for your help!

Hi Valerie,

that the probe detects the ladder happens oftener even whenn you use radioactive labeled probes, sometimes helpfull in my opinion. The thing is that your probe detects also wt DNA which should not happen. And in the lanes of interrest you get not an additional band in another size which could be your correct. What also look strange, is that lane 3 and 8 look different although the same DNA is loaded.

Maybe you can try to ilsolate your probe again and do a blot just positive and negative control DNA and try different ways of pre-hybridisation, hybridisation temperatures and washing temperatures and washing buffers.

For radioactive labelling I used the Prime-It RAndom Primer Labeling kit.

There you can also have this kind of problems. I prefer this method for looking for knock out bands and single copy genes, because it is more sensitive.

Best wishes

Sibylle