Miniprep Madness

jayni76 at my-deja.com jayni76 at my-deja.com
Wed Nov 1 10:13:43 EST 2000


Please help?? Suddenly, after having done spin minipreps for a long time, and
always getting excellent eluted concentrations ie. 500-1000ng/ul, I am
getting very low yields?? I have carried out the tests to see where the DNA
is going and it seems to be not binding to the spin column, but carrying
straight through with the rest of the lysate!! I have tried 3 different
qiagen kits, and a sigma kit, I have changed culture volume 2-5ml and doubled
P1, P2 and N3.	I have streaked glycerol stocks of plasmids that worked fine
1 month ago and that gave the same low yield, 10-100ng/ul.  I use differetnt
strains, JM109, XL1 Blue, Nova blue, DH5a, and they are making no difference!
It is making my job very difficult, as midipreps take a lot longer!! But they
are always good yields. Why can things change so suddenly??  The only
difference I can see is that lysis doesn't look too perfect and the
neutralisation step looks a little too much like egg white, gluupy rather
than fluffy, and when spun it forms a very compact pellet. Has anybody any
ideas as I have totally run out!!

Thanks
Jayne


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