Product Support – Protocols

Cy5 Thermo Sequenase Dye Terminator Kit
(Product Code 27-2682-01, Amersham Pharmacia Biotech.)

Revised Protocol for use with the MicroGene Clipper™ or Long-Read Tower™ Sequencer

The following DNA templates were tested using the Cy5 Thermo Sequenase Dye Terminator Kit:

200 ng DNA (m13mp18),
1.5 µg pBS with 1.4kb insert,
1.0 µg pUC control provided with the Cy5 kit,
1.5 µg pBS clone with a high GC content,
200 ng PCR product (1.3 kb)

  1. Remove all kit reagents from the freezer (except enzyme) and thaw on ice. Vortex gently, microfuge each tube and place on ice. Remove enzyme from freezer just before use and return immediately after use.
  2. Prepare stock d/dd termination mixes according to ‘Procedure A’ in the protocol booklet supplied in the kit. Label four 0.2 mL thin walled tubes ‘A’, ‘C’, ‘G’ and ‘T’ and place on ice. Aliquot 2 µL each of the A, C, G, T d/dd termination mixes into their labeled tubes respectively.
  3. In a separate 0.5 mL tube, add the following to make a master mix:
Primer* (2-5 pmol/µL) 2.0 µL
Reaction Buffer 3.5 µL
Thermo Sequenase enzyme (10U/µL) 1.0 µL
DNA + dH2O 20.5 µL
Total volume 27.0 µL
  1. Aliquot 6 µL of the above mixture into each of the termination tubes. Pipette gently up and down during each step. Place into a pre-heated thermocycler and immediately begin the following program.(It is recommended to use a thermocycler with a heated lid. If a heated lid is not a part of your instrument, top samples with a small amount (10 µL) of light mineral oil to prevent evaporation.)

    * When using custom primers, it may be necessary to increase the primer quantity up to 10 pmol / reaction. Cycling Conditions
    The following cycling program was performed on an MJ PTC200 thermocycler. As different thermocyclers may vary, please ensure the ramp time is adjusted to 1°C/sec.

    Initial Denaturation 95°C 2min cycle 1x
    Denaturation 95°C 30sec
    Annealing 60°C* 30sec cycle 30x
    Extension 72°C 80 sec
    Final Extension 72°C 5 min cycle 1x
    Hold 4°C

    * Annealing temperature of control M13 modified primer supplied in kit. The annealing temperature for custom primers must be determined empirically.

    General Notes

    • After cycling, DNA sequencing products can be cleaned using the ammonium-acetate/glycogen precipitation method or by G50 columns as described in the protocol booklet. If precipitation is preferred, we found that precipitating on dry ice (20 min) or -70°C (1 hour) yield very good results.
    • The centrifugation step performed at 4°C is highly recommended.
    • Extreme care must be taken as it is very difficult to visualize the precipitated DNA pellet. Avoid displacing pellet by high vacuum or by pipetting during the wash steps.

    Sample Loading
    All reactions are resuspended in 5 µL pink* Stop Loading Dye, heated for 2 min at 75°C and place on ice. Mix thoroughly by pipetting up and down and load 2µL into each well of the MicroCel™ cassette.

    Important:
    Do not use the dextran blue loading dye supplied in the kit. This dye will quench the signal intensity when samples are run on the OpenGene™ System.
    Please contact Visible Genetics Inc. (1.888.463.6844) for further information on obtaining the correct loading dye.

    Summary
    All reactions performed using the Cy5 dye terminator kit gave good sequences. These results were compared to those generated with the Cy5.5 dye terminator kit. The results were equivalent: very low background and good peak signals. Both clean-up methods (either Ammonium-Acetate/Glycogen precipitation or G50 columns) worked well. Tips outlined in the general notes above are highly recommended.