southern blot of plant genomic dna - no band detected! percentage of homology matter? (Jan/13/2008 )
lowering the stringency on your hybridisation is OK, at least to give you a band... however, if you are getting good signal in your positive, I suspect that the problem is with either the DNA or the sequence you are using. Purity of the DNA is important, try using a Qiagen kit for isolation, they are pretty good, though a little pricey.
How did you design your probe? is the PCR based on a gene from another species or from a cloned gene from your target species? if you are using another species I would worry that there is some sequence difference that is not allowing your probe to bind.
sorry for the late reply
thank you very much for your suggestion rodpck and bob1,
i tried at 37 degree gave the same result, only positive control , but strangely there is less non specific binding with the marker, its a much cleaner blot. i had reduced the washing temp and also temp.
the probe was designed from restriction digested DNA from plasmid containing the gene fragment. and ya i think there is some , though less than 5% difference between the cloned fragment and the species that i am trying to hybridize with. though not in the case of the transgenic plant, it should be more similar, but dont know about the copy number.
in that case if i use less stringency shouldnt it help?
i read in a practical manual that if the degree of similarity is less and in case of double stranded probes keeping for longer time will hurt the hybridization .
and the salt concentration will also affect the binding. any thoughts?
and i think purity of DNA might also be a problem, wil use Qiagen next time. and reduce the probe lenght too.
i am trying to fix a temperature and time for the hybridization and the washing steps. these seems to be the steps that seems to need more attention.
Dear phytoviridae/ laxmi,
i wish u might have already figured out the solution, if not some suggestions from my side.
From the entire discusion i did'nt get few points, i.e whether ur gene is a native gene of tobacco or u want to see is there any homologs of ur gene of intrest in tobacco.
if it is a native gene, generally u should get it stright away in southern, If ur looking for homologs of ur gene in tobacco and u take a probe belonging to the UTR of ur gene may be u wont have enough homology at the UTR level to get the signal. this is one possibility.
u mentioned u raised transgenic with a partial gene, if u confirm it with genomic PCr ( with marker gene amplification)before going for southern.
try some other enzymes as well for digestion. most of the common enzymes will work well with nicotiana...
all the best ...
Thank you gene_tag,
i am still searching for an answer, i did a DNA dot blot with the PCR product from the plant to the CDNA of the gene and PCR of the gene, the result was very good, but no binding in the southern. i wonder if this result will be acceptable when going for publication.
You are right in saying that i a searching for a homolog of the gene, the CDNA I have is not 100% similiar to the gene in the plant.
but we got very good PCR from both transgene and wild type plant. but i diddnt detect the band even in the transgene that shoul d have the 100 % similar gene construct too.
is there any other way we can detect the non homolog gene in plant?
any suggestion appreciated.
it all depends what journal you want to publish into. i hope you have already solved this issue. it really sucks when you spend a lot of time trying to figure something out and it just doesn't come out well. have you tried sequencing of the fragment that is not 100% specific (in case you're getting a product by pcr you could clone and sequence)? i certainly hope you already got your southern blot and everything is going well.
thank you toejam for ur reply
i have given up on southern blot and going ahead with dot blot ,
about the sequencing i did the sequencing of the PCR product,