agarose gel electrophoresis - fluorescence which will not migrate from well and does not disappear a (Aug/11/2010 )
I have carried out a phage nucleic acid extraction and when I run the sample on an agarose gel stained with ethidium bromide, fluorescence can be seen in the well. However, it will not migrate even if the gel is run for 2hrs.
In addition, to gain more information on the nucleic acid composition of the phage genome I digested the sample with both RNA and DNA and in both cases fluorescence could still be seen in the well.
I am now questioning if my extraction procedure worked at all and if the ethidium bromide has not just bound to some other contaminant in the sample.
What could ethidium bromide bind to other than nucleic acid? Thanks for any suggestions and comments.
keary on Wed Aug 11 14:42:22 2010 said:
I have carried out a phage nucleic acid extraction and when I run the sample on an agarose gel stained with ethidium bromide, fluorescence can be seen in the well. However, it will not migrate even if the gel is run for 2hrs.
In addition, to gain more information on the nucleic acid composition of the phage genome I digested the sample with both RNA and DNA and in both cases fluorescence could still be seen in the well.
I am now questioning if my extraction procedure worked at all and if the ethidium bromide has not just bound to some other contaminant in the sample.
What could ethidium bromide bind to other than nucleic acid? Thanks for any suggestions and comments.
Hi,
First thing I would ask is that what's the concentration of your agarose gel? Too high percentage might result to the nucleic acid not migrating despite of long running time. Also what's the voltage you are running the gel on?
Have you tried to determine the nucleic acid concentration of the extract? If it shows a good peak at Abs. 260nm then it probably indicates that there are nucleic acids present and you don't really need to worry about your extraction protocol.
Hope that helps
Hi,
I was using the lowest precentage I belive you can 0.5%. Also I measured samples using nanodrop and got nice peak at 260 and concentrations up to 1000ng/ul.
I am just after reading that salt concentrations inhibit DNAase activity. My bacteriophage were propagated in sea water and were precipitated with zinc chloride so maybe this prevented DNAase from breaking down the DNA (if thats what it is). What do you think? Would the salt have escaped the phenol chloroform extraction and ethanol precpitation?
Thanks for feedback 
PS gel was small so I was using 90V
if you use the TBE buffer it moves very slow may be if youuse TAE buffer it might move faster
ranvi on Wed Aug 11 17:45:22 2010 said:
if you use the TBE buffer it moves very slow may be if youuse TAE buffer it might move faster
Yes I always use TAE buffer for my electrophoresis.
Is there any chance that anything else other than nucleic acid is staining?