DNA isolation troubleshoot. - (Apr/13/2007 )
We've been isolating gDNA from either total blood samples or liver tissue from sheep using the QIAGEN DNeasy Blood & Tissue Kit.
The liver tissue samples are for confirmation of primer design, and the collection date is unknown.
Nanodrop readings indicate good OD ratios, good concentration per uL, in general a good curve. BUT when we run our samples in a gel, we find that they appear to be degraded. Yet, within the continuous smear some clear bands are visible for all samples (25) and all of them occur equally for all samples (ie. all same sizes).
We take upmost care of procedures, it is almost like some restriction enzyme would have been added. We don't think there would be any nuclease contamination and if it was because of mechanical shearing the fragments would be random and not equals.
If anybody has any idea why this can happen I would aprecciate your comments.
Could it be a contamination in the electrophoresis buffer (PCR product,...). Degradation is not so likely to form equal bands. Did you try any downstream application, yet?
hmmmm... could be that, but I don't see how the stock buffer could be contaminated. We'll run a PCR on Monday to see what happens. The funny thing is that we do lots of extractions and randomly this occurs, and as I said before, the odd thing is the bands in the smear.
Thanks for your help