BAC DNA prep for end sequencing

Laura F Marek lmarek at iastate.edu
Wed Mar 10 10:29:44 EST 1999


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
>ng 
>for me, and I suspect there is some finesse beyond that required for 
>ordinary-sized plasmids. Thanks for any help. 
>
>Nancy Phillips
>phillinj at slu.edu
>Department of Pathology
>St. Louis University Hospital
>3635 Vista Ave.
>St. Louis, MO, 63110, USA
>ph:(314)577-8782
>fax:(314)268-5120
>
>ph
Nancy,

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
Agronomy Department
Iowa State University




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