plasmid DNA isolation - problem in plasmid DNA isolation (Jun/29/2005 )

Hi all,
i am having a very surprising problem
i am trying to purify DNA from DH5a cells by alkaline lysis method and getting a smear from the well to the plasmid and no smear down the plasmid
most probably they are proteins
i tried changing the phenol, potassium acetate but all in vain
this could be looking very silly but this is troubling me since months
and i am not able to digest the plasmid for my subsequent cloning purposes
any helpful suggestions would be grateful
leelaram

This sounds like contamination with chromosomal DNA. After adding Solution II, invert the tube gently until the mixture is clear, generally 5-7 times. Do not vortex mixtures containing Solution II, as this will shear chromosomal DNA which will then contaminate the plasmid prep.

[quote=tfitzwater,Jun 29 2005, 11:21 AM]
This sounds like contamination with chromosomal DNA. After adding Solution II, invert the tube gently until the mixture is clear, generally 5-7 times. Do not vortex mixtures containing Solution II, as this will shear chromosomal DNA which will then contaminate the plasmid prep.

Hi,
thanks for the suggestion
but if at all it is chromosomal DNA isolation why is it a smear only upto the
plasmid, whichever it is, i am trying to isolate
rather, i would form a smear upto the lower dye

Chromosomal DNA generally runs as a tight band at about 23kb (if it is not degraded).

Try doing a restriction digest on this prep and run it on a gel compared to undigested. If it is chromosomal contamination you will see a difference... you will also have demonstrated a DNAse contamination in your prep (the smeared DNA).