Hi Yasser,

Looks like all of your probe is going and binding to the ladder. I have had that issue with some of the probes I have used. I would suggest cutting off the ladder from the blot before hybridization that might increase the chances of your probe binding to your digested DNA. Also you can try to digest your DNA wiith other enzymes as well and see if this robe gives you the sizes you want. I have used the automated southern blot probe design tool listed in this publication

http://www.biomedcentral.com/1471-2164/11/74

Good luck

Priya

Hi Yasser,

Looks like all of your probe is going and binding to the ladder. I have had that issue with some of the probes I have used. I would suggest cutting off the ladder from the blot before hybridization that might increase the chances of your probe binding to your digested DNA. Also you can try to digest your DNA wiith other enzymes as well and see if this robe gives you the sizes you want. I have used the automated southern blot probe design tool listed in this publication

http://www.biomedcentral.com/1471-2164/11/74

Good luck

Priya

Hi Priya

Thanks a lot for your answer and suggestion but unfortunately i tried it before.

i will try the automated southern blot probe design tool to check my probe accuracy

Thanks

Yasser

Hi Yasser,

how did you prepare your probe? Probes prepared by RE cutting of plasmid vectors could produce ladder hybridization due to vector contamination. If so, you can try PCR amplification of your probe .

You can also increase the amount of DNA transferred on the membrane.

Good luck

HI Yaseer

Basically PCR based detection and real-time based detection amplifes a sequence several folds ...and detection is easy...by southern analysis (or northern analysis) you are detecting a gene using onle a fix amount of DNA/RNA.....Many times if a gene is very low copy (low expressed) it is difficult to detect it by blotting technique......You may have to either increase the amount of nucleic acid....if still not possible...the results based of RT-PCR should be fine...

Besides...which detection method are you using for detection in Blotting...?

bye

1st question, how do you know you have transfered all digested DNA? I am not sure what protocol you are following, basically, we denature double strand DNA in gels with NaOH or HCl, after this step, you can not see any EtBr stained DNA. but since your ladder coulod be detected, it looks like you did at step.

2nd question could you upload the gel first? we can see more details.

let's talk more details after taking a look at the pictures, first.

good luck

You can labeled the PCR product and use it as a probe. Also use as a positive control , PCR product in one line of the gel in order to test that all is going well. If you have more troubles, I believe more DNA must be used in the digestion proces or the probe that you use is not correct. Have you sequenced the fragment you use as a probe?. Do you know the DNA sequence of the gene you are looking for?.

I used to do strand specific DNA repair southerns with a >20kb restriction enzyme fragment. At first, I had similar issues as you. The problem I had was I was too violent with the DNA in processing, vortexing in particular. For large targets you can easily mechanically shear the DNA at random so the band will not form. The results from this issue is great ladder and no bands of target gene. If you have a target band

I had this problem a couple of years ago but I find that temperature of oven hybridization isn’t true. I think that you must check temperature inside oven with indicator of set.

Best wishes

Nader

Hi!

I wonder how do you confirm by seeing the blot that digested DNA is transferred.

Had it been gel once the transfer is complete you can check the gel with UV transilluminator so that you can say confidently whether the transfer is complete or not. Now I doubt whether the transfer is complete or not.

You might have followed prehyb. How was your labeling, and count , hope you might have done hybridization for o/n with correct /optimum temp, (if you check the bolt with counter then it gives max. noise, do not think that your transfer/hybridization is complete). Followed by careful washing, with different stringency. If everything is done properly it should come. Do you have any gel picture of your digestion or some thing?

Dear all

thanks a lot for your valuable information about southern blot, i will summarize my steps and upload gel pic as soon as possible but i will try to be clear in my question

1- i used Fermentas* Biotin Chromogenic Detection Kit in the detection and follow the protocol without modification

2- i checked the gel after transfer and found no traces of any band and there is a little ethidium in the rest of the gel, and sometimes i checked the membrane using UV transilluminator and found the digestion and the ladder

3- i used pcr to prepare my probe

sorry for being late to make it clear for you but i have some issues

thanks again

Yasser Morsy

for 2, if you are working on norther blot (RNA), the method is right (EtBR staining on membrane and gel).

but on southern blot, you need to denature the double strand DNA into single strand one, so, after denaturation, even you put the gel under UV, you can not see any band. Do you do denaturation and neutralization? if not, it will be the problem.

Also DNA quality is the key. Make sure you have clean high molecular weight DNA that is digested well by restriction enzymes. If your DNA is low quality, you won't have a nice band.

I would recommend you to amplify your gene of interest by PCR and then clone it into vecor. After getting your template DNA from vector for probe making, it would enhance your template binding specificity and you could see your nice bands. You should better not use the PCR product for southern probe, it can give your non-specific bands.