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œœœœœ@SEQUENCING DOUBLE-STRANDED DNA@œœœœœ



@A variety of sequencing procedures are available - in our hands, the best of these is double-strand sequencing using sequenase and 7-deaza-dGTP. The method is easy and reliable, consistently giving 300 nucleotides of clear, clean readable sequence. Unlike many dsDNA sequencing procedures, supercoiled DNA is used. In fact, miniprep DNA isolated using NaOH-SDS procedures works as well as CsCl-purified DNA. The procedure is, however, quite sensitive to the host-strain of E.coli - DH5alpha works very well, whereas the JM-series work poorly. With CsCl-purified DNA, of course, the host strain doesn't matter. Deaza-dGTP is used to help prevent band compression caused by secondary structure in the DNA product - although not always needed, the sequences are as readable as those using dGTP so we use deaza-dGTP all the time. dITP is not recommended. Sequencing reactions are easiest done 6 at a time (one set of gels worth).

  1. SEQUENCING REAGENTS

    1. STOCKS:
      @10mM dNTPs
      @10.6 mM 7-deaza-dGTP (USB #70065)
      @10mM ddNTPs

    2. Labeling mix:
      per ml 10X final conc.
      10mM 7-deaza-dGTP 1.5ul 1.5uM
      10mM dCTP 1.5ul 1.5uM
      10mM dTTP 1.5ul 1.5uM
      dH2O 995.5ul
      store 1X & 10X stocks in 100ul aliquots at -20C. use 1X stock for sequencing reactions.

    3. Termination mixes:
      (final conc: 147uM dATP/dTTP/dCTP/dGTP, 50mM NaCl, 2.7uM ddNTP)

    4. solution #1: (standard termination reaction mixes)
      ddATP ddGTP ddCTP ddTTP
      10mM 7-deaza-dGTP 8 8 8 8
      10mM dATP 8 8 8 8
      10mM dTTP 8 8 8 8
      10mM dCTP 8 8 8 8
      5M NaCl 10 10 10 10
      10mM ddNTP 0.8ddA 0.8ddG 0.8ddC 0.8ddT
      dH2O 957 957 957 957
      for 1ml (volume in ul)

    5. solution #2: ("extending mix")
      10mM 7-deaza-dGTP 18
      10mM dATP 18
      10mM dTTP 18
      10mM dCTP 18
      5M NaCl 10
      dH2O 918
      for 1ml (volume in ul)

    6. combine 2 parts solution #2 to 1 part of each solution #1
      store in 100ul aliquots at -20C.

    7. Annealing mix (Rx buffer):
      @ per ml(1X) final conc
      1M Tris-Cl, pH 7.5 200ul 200mM
      1M MgCl2 100ul 100mM
      5M NaCl 50ul 250mM
      dH2O 650ul @

    8. Gel sample buffer
      per 10ml final conc.
      formamide 9.5ml 95%
      500mM EDTA 0.4ml 20mM
      bromophenol blue 5mg 0.05%
      xylene cyanol FF 5mg 0.05%

    9. 35S-dATP
      @alpha-35S-thio-dATP at about 1000Ci/mmol, 10uCi/ul

    10. Sequencing primers
      @gel or FPLC-purified oligonucleotides at 0.5 pmol/ul (~2.5ng/ul)

  2. SEQUENASE SEQUENCING PROCEDURE

    1. RNase treatment: (skip if samples are already RNased or CsCl)
      Combine: 2.5ul miniprep DNA (~1ug)
      6.5ul ddH2O
      1.0ul 100ug/ml RNase A
      ------------------------------> 10 min. RT (room temperature)

    2. Denaturation:
      Add: 10ul 400mM NaOH
      ------------------------------> 5 min. RT
      Add: 3ul 2M NH4OAc pH 4.5
      9ul ddH2O
      75ul 95% EtOH
      ------------------------------> 10 min. in dry ice:EtOH bath
      spin 10 min in microfuge, decant sup
      fill the tube with -20C 70% EtOH, decant & invert over a paper towel. Tap dry, then dry in speed vac.

    3. Sequencing:
      Add to dried denatured DNA
      2ul reaction buffer
      8ul 0.5 pmol/ul primer
      ------------------------------> 10 min. 37C
      (Use this incubation time to prepare labeling reaction pre-mix and aliquot 3ul termination mixes into each of 4 tubes per reaction.)

    4. Prepare labeling reaction pre-mix:
      1ul 100mM DTT for 6 tubes: 6.5ul 100mM DTT
      2ul 1X labeling mix 13.0ul labeling mix
      0.7ul ddH2O 4.6ul ddH2O
      1ul 35S-dATP 6.0ul 35S-dATP
      0.3ul Sequenase 2.0ul Sequenase

      Add 5.0ul labeling reaction pre-mix per tube
      ----------------------------> 2-5 min. RT

    5. Termination Reactions:

      Add 3.5ul of each reaction to 4 tubes containing 3ul of each dideoxy termination mix.
      ------------------------------> 10 min. 37C

    6. Dry in speed-vac

      Add 10ul gel sample buffer --> vortex (optional: store -20C)

  3. SEQUENCING GEL

    1. Prerun sequencing gels for 30 min at 1200 volts.
    2. Heat the samples to 90C for 2 min, and quick-cool on ice before loading.
    3. Run 1-2ul aliquots on sequencing gels at 1300-1500 volts (typical gels are 6% polyacrylamide-bis 50% w/v urea) Be careful not to run the gels too hot!
    4. For "short" runs, run the xylene cyanol 2/3 to 3/4 of the way down the gel. For long runs; when the xylene cyanol is 3/4 of the way down the gel on the initial loading, load 'blank' sample buffer & run until this second loading of xylene cyanol is 3/4 of the way down the gel.
    5. Soak the gel for 15 min. in 10% MeOH, 10% acetic acid, 2% glycerol, lift to 3MM paper & dry in gel drier for 30 min. at 80C.
    6. Autoradiograph with the film in DIRECT contact with the dried gel overnight & develop.


USB Sequenase protocol
USB 7-deaza-dGTP sequencing protocol
ESHaas, PaceLabs

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