Help with DNA precipitation - (Jul/03/2007 )
Hi guys,
I am attempting to isolate DNA from BAL (bronchio Aveolar Lavage) by isopropanol precipitation. Basically all that is, is a saline wash of a person's lung, to clear exces proteins, debris and such (it's the current treatment for PAP patients, ones who have accumulation of protein in the lung). For some reason, straight precipitation with no lysis step to remove any debris or proteins seems to yield a higher amount of DNA (150 mM salt is present). Also manganese seems to inhibit precipitation of DNA (lower yield).
Can anyone explain this?
-jalin-
QUOTE (jalin @ Jul 3 2007, 10:54 AM)
Hi guys,
I am attempting to isolate DNA from BAL (bronchio Aveolar Lavage) by isopropanol precipitation. Basically all that is, is a saline wash of a person's lung, to clear exces proteins, debris and such (it's the current treatment for PAP patients, ones who have accumulation of protein in the lung). For some reason, straight precipitation with no lysis step to remove any debris or proteins seems to yield a higher amount of DNA (150 mM salt is present). Also manganese seems to inhibit precipitation of DNA (lower yield).
Can anyone explain this?
I am attempting to isolate DNA from BAL (bronchio Aveolar Lavage) by isopropanol precipitation. Basically all that is, is a saline wash of a person's lung, to clear exces proteins, debris and such (it's the current treatment for PAP patients, ones who have accumulation of protein in the lung). For some reason, straight precipitation with no lysis step to remove any debris or proteins seems to yield a higher amount of DNA (150 mM salt is present). Also manganese seems to inhibit precipitation of DNA (lower yield).
Can anyone explain this?
hi jalin,
that seems strange especially since you would be lavaging a lot of alveolar macrophages, that would contribute to the total DNA if they were lysed. i guess that one possibility is that the alveolar proteins are associated with the DNA. if this was the case you would get worse 260/280 ratios in the first method compared to a cleaner method such as removing the debris.
-mateo-