What could be the reason for not seeing any bands for plasmid on gel? the dna amount is 200 ng which should be enough, 5 ul 1% ethedium bromide which is also more than enough. I see weak bands for the ladder but none for the plasmid. I am trying to determine its purity but im not seeing anything. Could it be agarose because it has been sitting on shelf for quite a while?
-fred1972-
Sounds like either your restriction enzyme, it's buffer, BSA or your H2O or pipettes are contaminated with DNAse...
Agarose keeps for ages.
QUOTE (fred1972 @ Sep 14 2007, 02:17 PM)
What could be the reason for not seeing any bands for plasmid on gel? the dna amount is 200 ng which should be enough, 5 ul 1% ethedium bromide which is also more than enough. I see weak bands for the ladder but none for the plasmid. I am trying to determine its purity but im not seeing anything. Could it be agarose because it has been sitting on shelf for quite a while?
-LeserattePD-
But from where? i use different sterile pipettes. The water is just sterile distilled water and not dnanuclease free but how would water be contaminated from dnase and from where?
QUOTE (LeserattePD @ Sep 14 2007, 10:13 AM)
Sounds like either your restriction enzyme, it's buffer, BSA or your H2O or pipettes are contaminated with DNAse...
Agarose keeps for ages.
QUOTE (fred1972 @ Sep 14 2007, 02:17 PM)
What could be the reason for not seeing any bands for plasmid on gel? the dna amount is 200 ng which should be enough, 5 ul 1% ethedium bromide which is also more than enough. I see weak bands for the ladder but none for the plasmid. I am trying to determine its purity but im not seeing anything. Could it be agarose because it has been sitting on shelf for quite a while?
-fred1972-
AHA! Found it i was using water instead of TAE buffer DUH 
QUOTE (fred1972 @ Sep 14 2007, 09:25 AM)
But from where? i use different sterile pipettes. The water is just sterile distilled water and not dnanuclease free but how would water be contaminated from dnase and from where?
QUOTE (LeserattePD @ Sep 14 2007, 10:13 AM)
Sounds like either your restriction enzyme, it's buffer, BSA or your H2O or pipettes are contaminated with DNAse...
Agarose keeps for ages.
QUOTE (fred1972 @ Sep 14 2007, 02:17 PM)
What could be the reason for not seeing any bands for plasmid on gel? the dna amount is 200 ng which should be enough, 5 ul 1% ethedium bromide which is also more than enough. I see weak bands for the ladder but none for the plasmid. I am trying to determine its purity but im not seeing anything. Could it be agarose because it has been sitting on shelf for quite a while?
-fred1972-
