miniprep problem - (Jul/23/2005 )

I am doing this mniprep for my ligation mix using dh5alpha and XL1blue competent cells.I am doing miniprep following Maniatis alkaline lysis method. But consistently I have been getting a thick viscous thing and a pellet which is not ready to dissolve in H2O with RNase. In the gel I could see a degraded DNA smear(attachment, middle lane is earlier miniprep of only vector )I tried changing all the reagents except for the comp cells. Still the result is same!! What could be the reason... Plz advise!!

Some E.coli strains under some growth conditions accumulate large amounts of polysaccharides which then co-purify with the DNA. I suggest using a different strain.

I have also found that this is less of a problem if the cells don't enter lag phase. The easiest way to do this is grow at 30C rather than 37C o/n.

hi
if the central band is a previous prep, i would sa you didn't purify anything in the other lanes...
I think Daniel is correct by saying log phase is the first critical point in plasmid preps.
If you think your strain is a problem, try to see if you can get help from a neighbourg lab.
Fred