John M. S. Bartlett.pdf - Bio-Nica.info

350 Kösel et al.
3. Methods
3.1. Purification of Sequencing Templates
1. PCR is performed according to established protocols (7). After PCR amplification, PCR
products are purified away from excess nucleotides and primers using either spin column
chromatography (Wizard PCR Preps DNA Purification System, Promega, Madison, WI)
or ultrafiltration (Microcon-30, Amicon). The purification systems are used according to
manufacturer’s recommendation (see Note 3).
2. Measure DNA concentration using the fluorometer and Hoechst dye No. 33258. The dye
is dissolved in 1× TNE (prepare two stocks, 0.1 and 1 µg/mL, respectively). Calf thymus
DNA (100 or 1000 ng/µL) is used as a standard for calibration (see Note 4).
3. Purification results may be checked by electrophoresis of samples through a 2% highmelting-point
agarose gel.
3.2. Sequencing Reactions
1. Sequencing reactions are set up in a total volume of 20 µL containing the follow: 13 µL of
DNA template solution (2–4 pmol); dilute with sterile, double-distilled water, if necessary;
3 µL of 5′-digoxigenin end-labeled primer (1 pmol/µL); 2 µL of 10× reaction buffer
(250 mM Tris-HCl, pH 8.0, 50 mM MgCl 2 ); and 2 µL of Taq polymerase (3 U/µL).
2. For each sequencing reaction, transfer 4 µL of the above mixture to four thin-walled PCR
tubes, each containing 2 µL of the respective termination mixture (ddATP, ddCTP, ddTTP,
and ddGTP; see Note 5).
3. Overlay samples with 20 µL of mineral oil, and centrifuge for a few seconds in an
Eppendorf tube, centrifuge at full speed.
4. Cycle sequencing is performed using a thermal cycling protocol empirically optimized for
the T m of the sequencing primer. When using sequencing primers of the same sequence
and length as those used for PCR, a thermal cycling protocol identical to the one used for
PCR usually gives good results (see Note 6).
3.3. Preparation of Sequencing Gel
For gel electrophoretic separation of sequencing reactions, an 8% denaturing
polyacrylamide gel and standard sequencing equipment are used.
1. Clean glass plates thoroughly with 70% ethanol. Cover inner surface of one plate with
a few drops or Sigmacote and let evaporate for 5 to 10 min. Set up glass plates with
spacers and seal edges airtight using Scotch electrical tape. Put two strong metal clamps
on each side.
2. Add 450 µL of 10% ammonium persulphate to one bottle (75 mL) of GelMix 8 and
mix gently. Slowly fill the space between the glass plates (avoid air bubbles!). Let gel
polymerize for at least 1 h at room temperature. (Wear protective gloves when handling
unpolymerized acrylamide.)
3. After polymerization of gel, remove clamps and electrical tape from the lower end of
the glass plates and transfer gel to sequencing apparatus. Fill the upper and lower buffer
chambers with 500 mL of 1× TBE each. Rinse sample wells of gel with 1× TBE using a
10-mL disposable syringe with needle.
3.4. Gel Electrophoresis and Visualization of Sequencing Results
3.4.1. Electrophoresis and Contact Blotting
1. Add 3 µL of loading buffer to each tube containing the sequencing reactions with the
respective “A,” “C,” “G,” and “T” termination mixtures. Centrifuge for a few seconds