Sequencing P1s, PACs or BACs

[Akira]

Dear Dr. Hall,

        We sequence P1, PAC or BAC, using dye terminator kit (ABI or
Amersham).  The success rate is ~80-90 %, so I don't think ours is the 
best
protocol.

         1) Culture in LB + antibiotics 200-500 ml
         2) DNA extraction by PI-100 (Kurabo, DNA extraction machine. 
Yield
is better than manual extraction for P1/PAC/BAC)
         3) RNase treatment (final 100 ug/ml)
         4) Precipitation in 10% PEG 8000 + 0.5M NaCl
         5) Phenol/chloroform/isoamyl (25:24:1) extraction
         6) Precipitation in Et.OH with 2M NH4OAc
         7) Rinse in 70% Et.OH
         8) Suspend pellet in TE 20-50 ul
         9) Check DNA concentration by restriction digestion and 
electrophoresis
        10) Use 1-2 ug per cycle sequencing reaction in typical 20-ul 
reaction

        I feel there is nothing particular in this protocol.  The most
important thing is the amount of DNA.  We can get a good chromatogram,
using 0.1 ug of cosmid DNA.  But, 0.2-0.3 ug of P1 DNA is not enough for 
P1
sequencing.  I discard OD meter for measuring DNA concentration of
P1/PAC/BAC.  It's not reliable.  The traditional way, electrophoresis of
digested DNA, is better than OD meter for estimation of P1 concentration. 
When we can see good band, almost sequences are successful.  We don't
sonicate DNA, just use circular/intact DNA.


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                Akira Tanigami, MD, PhD.
                National Cancer Center Research Institute
                Radiobiology Division
                5-1-1, Tsukiji, Chuoku
                Tokyo 104, JAPAN
                Tel. +81-3-3542-2511  ext.4752
                Fax. +81-3-3542-0688
                e-mail:atanigam at gan2.res.ncc.go.jp
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