I extracted DNA from several hundreds blood sample by promega kit , one sample among all the others show a two clear fluorescent bands migrate to the negative pole , those bands appear without any staining just uv 310nm irradiation as in the gel image below . the bands color are red to orange ,
Can anyone tell me what is this , why , any idea ,?
thank
In my opinion the two band could be attribuible to the presence of a proteic contaminants. Maybe the colour depends on the denaturation of proteins exposed at UV radiation.
Really interesting.
i do not think that the proteins have this ability of fluorescentation , even if it denature ,
Mr Atif Baig , i tested it under no filter and with ethidium bromide filter , both are gave a good fluorescentation but it is better under ethidium bromide filter .(this is for emission filter) , and i put the gel on 310 nm excitation filter .
Hello Hayder,
Could it be some porphyrin derivatives coming from heme decay from reticulocytes? A derivative of bilirubin?
Best regards
Prof Philippe Urban
Can it be BET (Ethydium bromide) contamination, showing only stronger bands.???
BET is very persistant, and if you use not properly cleaned cuve, it can stain your DNA...
Hope I did not misunderstood your problem, good luck.
I think that Davide Magrì is right to suggest that these are protein, which typically migrate to the negative pole (opposite DNA). There are several proteins that are fluorescent like gfp and all of its color variants. How fast do the bands move?
At this point, there is a problem. Proteins typically denature in the phenol solution common to DNA extraction kits. The denatured proteins should lose florescence.
http://www.ncbi.nlm.nih.gov/pubmed/19501614
This paper suggests that the gfp class of proteins do lose fluorescence when they denature but that some color variants are more resistant to denaturation depending on pH ... however, they did not not test resistance to phenol, which is very unkind to protein.
if the contamination is protein then it should have been destroyed in the extraction and lost during the filtering steps. This suggests that the contamination by a fluorescent protein occurred AFTER the extraction.
This is a cool puzzle. If you have more of that same blood sample try extracting it again and see if you get the same colored bands. This will tell you if the fluorescence is coming from the cell sample or a contaminated purification tube. Extract with another kit with a different batch number.
a re-purification of the contaminated DNA should also be clean from protein
(also check and clean the combs used to make the gel. Contamination on a bit of the comb could have introduced the protein.)
If these tests show fluorescence then your blood sample contains fluorescent markers that are probably NOT protein. What has the owner of this blood been up to? You could toss some of your DNA extraction on an poly-acrilamide gel and see what happens. I think contamination AFTER extraction is much more likely and you will answer that question with the experiments above.
I think EtBr contamination is unlikely as it seems to be moving in the same direction as the DNA and etbr move against the direction of the dna. . Where does gfp contamination come from...seems unlikely to be in the original tissue sample. Could it not be just a lot of RNA which has not been removed in the purification process?
Philippe Urban yes it could be . but they appear as two bands and i do not think that the agarose gel can resolve two kind of porphyrin derivatives . if they a porphyrin derivatives they have to be in single band .
thank
Benoit Maury : no i do not think so . because EtBr will migrate as a single band and it is more slowly than this
Joseph T Penrod: it is a human blood there is no way to be contain gfp or other flourescence engineered proteins .
second : i extract all the other samples with the same kit and there is no such bands in the DNA
Paul Rutland · : no they move toward the negative pole ie in the opposite direction of DNA and RNA movement , but those band could not be EtBr because
first the EtBr will diffuse quickly and would not appear as band
second : they are two well separated bands
Is it possible that the blood donor had recently had an angiogram or venogram of some type? Perhaps the fluorescin dye injected into the blood could retain some fluorescent properties.
Kimberly Susan Hall : yes i think this too , i will check the hospital database for this person
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