In article <36DB7F30.274E at sluvca.slu.edu>, phillinj at SLU.EDU says...
>>Hello, does anyone have a favorite miniprep protocol for BAC and PAC
>sequencing-quality DNA? So far, the alkaline lysis method is not worki
>for me, and I suspect there is some finesse beyond that required for
>ordinary-sized plasmids. Thanks for any help.
>phillinj at slu.edu>Department of Pathology
>St. Louis University Hospital
>3635 Vista Ave.
>St. Louis, MO, 63110, USA
When our sequencing facility was using rhodamine-based automated sequencing, my
alkaline lysis based BAC preps sequenced nicely 80-90% of the time. Since they
switched to the Big Dyes we had to change our DNA prep and ended up using Qiagn
midi-preps (tip 100s) with 100 mL cultures. Now they get great sequence 99% of
the time although usually an optimization run or two is required with each set
of preps to determine the appropriate concentration of template to use for the
sequencing reactions. We have just started working with the R.E.A.L. prep 96
system since we don't always need the amount of DNA produced with the midipreps
and we need to get more end sequences faster.
Laura Fredrick Marek
lmarek at iastate.edu
Iowa State University