Question
Asked 3rd Oct, 2012
  • Central Proteina Prima Company of Jakarta, Indonesia

How to design probe specific hybridize with a specific targeted gene in Southern blot method?

I use DIG-11-dUTP label probe with specific targeted gene to detect in host genome. Host genome have a huge sequence that to date no information in data base. I cut the genomic DNA by restriction enzymes but after southern blot process my specific gene in the host genome does not show the signal band. I use chemilluminence (SDP star) substrate for alkaline phospatase, I do not use radioactive label 32P. Could anyone give me more ideas with this case?

Most recent answer

21st Oct, 2012
Abdelhalim Boukaba
Chinese Academy of Sciences
Hi Heny! First do you see signal on non-digested genomic DNA. I guess you have also included this sample in you southern experiment. If yes, do you have a signal? Also how many bands you see with the digested plasmid?
Can you tell what your purpose to perform a southern blot on your digested DNA? Are you checking for proper targeted gene integration of your construct into the host genome? Is it for methylation analysis? Nucleosomes positioning? Etc… Do you know the pattern of your gene digestion in its genomic context?…With which specie you are working with and if possible do you know the copy number of your gene? You see, there are a lot of things to be considered before trying to find what went wrong and if there are better experimental alternatives to address your question.
I think your probe is OK from the signal you are seen with the plasmid and most likely also the hybridization conditions. For perfect match of your probe you should always use high strigency no matter how complex is your genomic DNA. I couldn’t download your gel picture…I can’t tell.
Just to discard any artifact with the southern blot, I suggest you can perform a slot blot or dot blot with digested and non-digested genomic DNA on the same membrane you use, include an alien genomic DNA as a negative control and your plasmid too. If you don’t have a vacuum manifold then do it manually by spotting 1-2 µl sample (I can’t tell you the concentration as I don’t know which specie you re working with and copy number of your gene) but try 5 to 10µg to ensure a good signal. Denature you DNA before applying it to the membrane and proceed with the rest as if you were doing southern, including UV-crosslinking.
Report back.

All Answers (10)

3rd Oct, 2012
Mohan Harikrishnan
Louisiana State University
Before going for hybridization check the quality and concentration of your probe. If you know the sequence details of your target gene along with UTR region, then you can use 3'UTR region as probe for hybridization. If you are using kit based detection check the troubleshooting guide to solve your problems.
4th Oct, 2012
Heny Budi Utari
Central Proteina Prima Company of Jakarta, Indonesia
I have checked the probe already, and I also have done for probe detection with different concentration, I used the high concentration that show positive signal. My restriction enzyme digested genomic DNA did not show a signal but my positive undigested plasmid show the band ( I load 20 ng/ul of plasmid positive)....could you give any suggestion to me...
5th Oct, 2012
Mohan Harikrishnan
Louisiana State University
Could you please post some gel pictures of your digested genomic dna and the blot. Maybe after seeing the picture we can get some ideas.
17th Oct, 2012
Meetul Kumar
Defence Research and Development Organisation
Digestion of the genomic DNA must be checked in gel. Specific labelling of transgene may be done using Ready -to-Go beads (Amersham) followed by detection through P32. The washing plays an important role. gives better results
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17th Oct, 2012
Heny Budi Utari
Central Proteina Prima Company of Jakarta, Indonesia
I send my agarose gel picture on the sample and the last right site that show the clear band is plasmid with the concentration 100 ng/ul, in the last repeated gel I use 20 ng/ul and was not show the band, but after hybridization with DIG-dUTP probe it will show the band, the other row of gel are my sample, but after hybridization process they were not show the expected band.....hope that you all help me, thanks
17th Oct, 2012
Heny Budi Utari
Central Proteina Prima Company of Jakarta, Indonesia
17th Oct, 2012
Mohan Harikrishnan
Louisiana State University
Initially you can try washing the blot with low stringency conditions and check for signals. Since plasmid DNA are small and intact they will produce good signal but its not the same with genomic DNA.
You can try the following steps. It may look silly but sometimes it will work better
1. Minimise the thickness of your agarose gel
2. Also you can try with SSPE buffer for transfer instead of SSC
2. UV-crosslink your blotting membrane before hybridisation for 2 mins
3. Optimise your DNA-DNA hybridisation temperature
4. Try low stringency washing.
17th Oct, 2012
Heny Budi Utari
Central Proteina Prima Company of Jakarta, Indonesia
OK I will try as your suggested, could you give me the method to detection with P32? So far I look around the labs in many univ. in thailand, they do not have that facility to do with radioactive, they change detected with chemilluminescence as I did. Could you recommended which abroad lab I should find and the professor or technisian I should contact?
17th Oct, 2012
Meetul Kumar
Defence Research and Development Organisation
try temp 65 degree for hybridization. also the labelled probe must be single strand, use PCR high temp 95 degree. low stringy wash is a good option. check the pH of the buffers that you are using. most important.
21st Oct, 2012
Abdelhalim Boukaba
Chinese Academy of Sciences
Hi Heny! First do you see signal on non-digested genomic DNA. I guess you have also included this sample in you southern experiment. If yes, do you have a signal? Also how many bands you see with the digested plasmid?
Can you tell what your purpose to perform a southern blot on your digested DNA? Are you checking for proper targeted gene integration of your construct into the host genome? Is it for methylation analysis? Nucleosomes positioning? Etc… Do you know the pattern of your gene digestion in its genomic context?…With which specie you are working with and if possible do you know the copy number of your gene? You see, there are a lot of things to be considered before trying to find what went wrong and if there are better experimental alternatives to address your question.
I think your probe is OK from the signal you are seen with the plasmid and most likely also the hybridization conditions. For perfect match of your probe you should always use high strigency no matter how complex is your genomic DNA. I couldn’t download your gel picture…I can’t tell.
Just to discard any artifact with the southern blot, I suggest you can perform a slot blot or dot blot with digested and non-digested genomic DNA on the same membrane you use, include an alien genomic DNA as a negative control and your plasmid too. If you don’t have a vacuum manifold then do it manually by spotting 1-2 µl sample (I can’t tell you the concentration as I don’t know which specie you re working with and copy number of your gene) but try 5 to 10µg to ensure a good signal. Denature you DNA before applying it to the membrane and proceed with the rest as if you were doing southern, including UV-crosslinking.
Report back.

Similar questions and discussions

How could I prevent the smear and unexpected bands in gel after restriction enzyme digestion?
Question
3 answers
  • Wenzel ChenWenzel Chen
Hello,
I performed a single digestion on my plasmids (~6.5kb) using BamHI. The total reaction volume was 10µl (containing 0.5µl(10U) BamHI) and the samples were incubated at 37° for 1hr.
After the digestion, I stored the digested samples at -20°C and run the DNA gel electrophoresis on the next day to check the samples. I used 1% agarose gel and 1X TAE buffer ,and I run the gel for 45min at 100V. (The dye was DL5000(6X) FluoroDye, smobio )
My expected result is: only 1 band should appear in lanes containing completely digested sample (~6.5kb but higher position because of its linear form)
However, the result of the gel electrophoresis (as the figure below) showed smear bands and some of the samples seems to be incompletely digested. In one of the digested samples, there are 2 bands on gel, one is obvious at 12kb and another is above 12kb , no 6kb band was shown.
I'm wondering why? Did my samples ligate with each other?
My questions are:
1) What may cause the smear band or the band shifting in gel?
I was thinking about the possibility of BamHI binding to DNA, DNA overloading, wrong RE digestion condition or electrophoresis condition...etc. Should I inactivate the enzyme by heating after digestion or before electrophoresis?
2) Why are my samples incompletely digested?
Did I set the reaction condition wrong?
Sorry for asking lots of question, and thank you so much for answering!

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