southern blot of plant genomic dna - no band detected! percentage of homology matter? (Jan/13/2008 )

hi all

please help !
been repeating this blot for months now!

i have been trying southern blot of genomic DNA from tobacco . I tried p32 labelling and DIG prob too.
but gives good result with positive control that is the plasmid with the gene fragment, but when nothing in the plant genomic dna

i Used 0.8% agarose gel for transfer -DNA high concentration more 20-30 microgram, used CTAB method for extraction.
Used both upward capillary movement in 10XSSC and electrophoresis in tank using 0.5X TBE
i checked the gel afterwards, the transfer was complete

transfer was done to nylon membrane
and uv crosslinked
washing in 2xssc
prehybrid-- company product--ambion ultrahyb and DIG easy hyb buffer-1 hour-- 42,55,65 degree celsius
Hybridization- overnight at various temperatures --42,55,65. degree celsius

washing
2XSSC +0.5% SDS
0.2Xssc +0.5% SDS
both 15 min 2 times at 42- 65 degree Celsius

p32 labelling was done at -70 overnight
and DIG with NBT BCIP overnight

all protocols gave good result in positive control
even dot blot ,
but nothing in the genomic dna

i had tried PCR from the genomic dna and got good bands, so the DNA must be there.
the probe for p32 was done using amersham beads and for DIG labelling was done using the DIG kit of roche,
the probe seems fine since positive control has very good binding , but thats since its 100% similar.

the probe i am using is only a small , less than 50% of the gene . the gene is not a known gene and only this part of the i have the sequence.
so could it be that this might be reason for difficult hybridization? but even in the transgenic plant (Checked by RT pCR and PCR and western, showed positive result) ?

should i try 32 or 25 degree ?
or is there something else i am missing?

please help, any suggestions ..

thank you in advance

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what are you digesting your DNA with? does this enzyme have a cut site in your probe?

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i am digesting it with EcoR1,and no there is no EcoR1 site in the probe?
does the digestion affect the probe?
i purify the DNA after the digestion with EtoH and sodium acetate and then wash with 70% etoh.

thanks for the reply though

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hi
how big is the tobbaco genome? i have done southern blots on maize genome (3.2 Mb approx) using 10 mg and it worked well. i have heard some other people using up to 50 mg and their blots were just a mess. maybe if you reduce the ammount of DNA you are using you could detect your bands. viel glück!!

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hi
thanks rodpck
did u mean microgram or milligram? i used microgram, i had tried 20microgram too, same effect, i am not sure of the size of tobacco genome.
i will try ur suggestion and see

thanks again

laxmi

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Dearest laxmi!

Unfortunately I'm not experienced on tobacco, so I can't tell you nothing on the technique or the protocol itself.

What I suggest you is to try with a genomic check of your hypothesis.
Which kind of gene is your interest gene: a transgene, a houskeeping gene, a resistance gene or else?

As you sequenced the gene on your own, maybe it worths to consider if your gene is conserved enough or if you can frequently find possible variations like alleles. Are you sure that in that in case of high eterozygositygene variations your probe will be able to attach anyway to the genomic DNA?
In order to have an idea about it, you can blast your sequence on the db. Did they sequence the complete genome of tobacco? What results do you obtain blasting the sequenced fragment of the gene? Does it blast on more than a conting?
Obviously, the importance of these information dependes on the type of samples you are analysing (clones of the same plant? different species?) and on the portion of the gene interested by the probe.

Did you try to make the hybridization of a wild type, well characterized gene (possibly presente in single copy) as a positive control? This can help you to discriminate if yours is a mere technical problem or not.

These are just suggestions, and probabily I didn't hit the point, but I hope that they may help you considering the problem from another point of view.
Let us know how you are doing.

ILA

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Dearest Ila

thank you very much for the suggestions. now i am more scared, i didnt take into account all these factors! actually it never entered my head to think like that

actually this gene has not been completely sequenced or i think not entered in the database.and blasting aganist tobacco genome ( whatever that has been sequenced ) didnt give any result, except for a few motifs similarity with rice and arabidopsis genome. though not the whole base pair.i think there could be alleles, in that case the hybridization wont be possible or difficult?


i dont know how conserved this gene is in various species either.Besides i believe this segment i am having is only a fragment of a bigger gene .i concluded that it was only a part of the gene since the western blot using antibody made against my fragment showed a very higher molecular weight band than what can be calculated from my fragment, and pcr and rt pcr showed similar sized band to my segment , but i have no idea how to get the full length.thats why i thought of doing southern .

i got good PCR from tobacco genome using primers specific to this fragment and sequenced it,, that showed high similarity with my sequenced segment. doesnt it prove that this fragment is there in the genome?

i had made transgenic tobacco plant with this part of the gene and tried to see if there is any difference in morphology or anything . THe pcr and rt pcr and even western showed the similar result when done with the wild type ,
though there is no binding to the probe in southern (made from this fragment )to the genome from the transgenic plant either, thats why i thought it could be the hybridization temperature or incubation time thats the problem, since theoretically atleast it should bind with the transgene in the transformed plant is int it?

so how does one go about finding the right hybzation temperature of the transgene in tobacco genome?
maybe if i solve that i can solve with the wild type.
i used the plasmid with the gene fragment as positive control, it showed good band in the southern , but not in the plant genomic dna.

any ideas?

laxmi

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You should be able to calculate the hybridisation temperature from the sequence of the probe, check your DIG manual for greater detail. If you are trying to clone/sequence the whole gene, then I would suggest doing RACE (rapid amplification of cDNA ends) to get the whole gene from RNA, I think Roche produces kits for it, but they are quite expensive.

10-20 ug of genomic DNA should work well. Are you sure that the DNA is being digested? EcoR1 is sensitive to supercoiling and CpG methylation, so if you don't have a smear on your gel you need to change your extraction proceedure to produce less supercoiled DNA. Alos check the quality of your DNA extraction, we have had problems with the DNA not being pure enough or being fragmented by the colums we were using to extract the DNA.

My comment earlier about the probe and cut sites was that... if you are digesting your genome with an enzyme and there is a cut site in the area where the probe would bind then the probe wouldn't bind as the site would be fragmented. Obviously there is no point in digesting the probe, but it is a common error in probe design.

How big is your probe? 100-500bp is ideal, much smaller or bigger than that runs into specificity problems and hybridiation problems respectively.

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Thank you Bob1 for your reply.

i had used 700bp probe and also 1.8Kb probe. both have same effect, and i calculated the tm from the DIG manual and tried that too, 44 degree only in positive control there is binding.

and there is no EcoR1 site in either of them.
the digestion is fine since i can see the good smear in the gel after purification and running in agarose.i used CTAB method for extraction so i think maybe there could be problem with that part maybe there is some chemical left in the extraction or purification that interferes with the hybrization or i have to further check more variations of hybridization temp.

if the full lenght gene and my fragment is less similar should i lower the temperature further?
anyone done hybridization at 25 or 37 degree?

and washing step also does reducing the temp give better result?

any suggestions welcome.

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hi phytoviridae,
i think your probes are really big, i used a 200 bp probe and it worked fine. i'm sorry about the mistake in my previous post, you're right it is 10 ug, not mg
i haven't tried hybridization at 25 or 37° or heard of anyone who has done this at my institute. apparently the tobacco genome is 4.5 mb, so, depending on the gene you're trying to detect (whether it is single copy gene, or multicopy, which you can not detect since the whole genome is not sequenced, but you can get a good idea depending on the hits you get, if any...) haha, just read that you already blasted and got a few motifs, what's the identity %? anyway, coming back to the main issue, 10 ug of DNA should be enough to detect single copy genes. try to reduce the size of your probe as bob1 suggests. the labeling with p32 should be fine. hope this helps.

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