biogeekgrrl at my-deja.com
Tue Oct 24 15:37:40 EST 2000
Yours is a common problem, don't fear. You've already lots of good
advice from the kind folks on this newsgroup. I'll just mention one or
two points which have, in my experience, proven to be important.
First, make sure you're getting complete lysis of your bacterial
pellet. If you have a 250ml overnight culture and spin all of it down,
try resuspending in twice the recommended amount of P1, and then double
the vols of P2 and P3. This has corrected the problem 85% of the time
in my experience.
Second, as has been mentioned, make sure you don't get any cell debris
in your Qiagen column. This generally mucks things up. I always run the
supernatant through a piece of gauze or filter paper before loading it
onto the column.
Other minor points: if you have a big plasmid ie 10kb-ish, these preps
are more troublesome. Try warming the elution buffer. Also, the pellet
after isopropanol precipitation tends to slip around the bottom of the
50ml tube. We always gently pipet off most of the isopropanol, then
transfer the pellet and remaining alcohol to a microfuge tube. It's
easier to remove the rest of the alcohol and do a 70% wash in these
Hope this helps.
"Like most scientists, Gabe was oblivious to the fact that no one gave
a rat's ass about research unless it could be expressed in terms of
Sent via Deja.com http://www.deja.com/
Before you buy.
More information about the Methods