230nm peak with NanoDrop - after gel purification (Jan/11/2009 )
hello there,
for a couple of months now we have huge problems with our gel purifications.
though dna is clearly visible on gel after cleanup, nanodrop gives only huge peak at 230nm, nothing else, picogreen doesn't work either
gel cleanup: qiagen kit
staining: etbr, sybrsafe, safeview - seems to not make a difference though first suspect
if the 230nm peak is organic solvents (phenol etc.) it should be there after column cleanup and elution in h2o or elution buffer
can't have to do with the stain? if stain would heavily incorporate into DNA, that would give peak at 312nm not 230nm, right?
really starting to get desparate here...
:-(
for a couple of months now we have huge problems with our gel purifications.
though dna is clearly visible on gel after cleanup, nanodrop gives only huge peak at 230nm, nothing else, picogreen doesn't work either
gel cleanup: qiagen kit
staining: etbr, sybrsafe, safeview - seems to not make a difference though first suspect
if the 230nm peak is organic solvents (phenol etc.) it should be there after column cleanup and elution in h2o or elution buffer
can't have to do with the stain? if stain would heavily incorporate into DNA, that would give peak at 312nm not 230nm, right?
really starting to get desparate here...
:-(
Normally it's the indication of chaostrophic salt contamination. The stuff used to bind DNA to the column.
TO fix this you could do an ethanol wash. That normally will get this problem fixed.
Althernatively u could prolong or increase the washing step in your gel extraction.
HOwever in my experience the extra ethanol precipiation + 70% wash is typically the best way.
And remember after the wash(es) to empty the collection tube, then spin the columns for a minute or two to remove residual wash solution and dry the columns before elution.