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Method: Sequencing of Double Stranded DNA Using Dideoxy Chain Termination

May 10, 1990. Updated June 4, 1997

Terry C. Lairmore


Principle:

Time required:

Special reagents:

All of the following required reagents are included in the SequenaseR kit (USB):

Special Equipment:

Description of Reactions:

Procedure:

Day 1

Day 2

    Remove the sequencing gel from refrigerator, place on Baserunner (IBI), and attach buffer chambers. After filling the buffer chambers with 1X TBE, gently flush out the wells with a syringe and needle and prerun for 30 minutes to 1 hour.

  1. Thaw the sequencing reaction samples, heat to 75-80 degrees C for 2 minutes and immediately load 1-3 Ál from per lane. Use the specialized microcapillary flat pipet tips (Midwest Scientific, #MPT1000F) to load the gel. Slowly layer each sample into the bottom of each well, avoiding bubbles.
  2. The lanes should be loaded G, A, T, C in adjacent order for each sequencing reaction. Run at a constant power of 55 watts, with voltage from 1800 to 2000 V or greater. A second aliquot of the samples may be heated again and reloaded after a variable period of time in order to read further. Ideally, an area of overlap between the bottom of the first load (larger fragments which have run longer) and the top of the second load (smaller fragments which have run less time) will be found on the autoradiogram. (One recommendation is to run the first load twice the amount of time it takes the bromophenol blue to run off, and the second load 2-2.5 hrs).
  3. At the completion of the run, turn off power supply, carefully drain buffer chambers (buffer must go in liquid radioactive waste container), and remove the gel from the apparatus.
  4. Place the gel with the glass plate face down on a flat surface. Remove the mirrored glass plate (the gel should stick to the unsiliconized glass plate). Leave the spacers in place on each side.
  5. Carefully soak the gel for 10-15 minutes in 10% glacial acetic acid on the glass plate with minimal rocking (be careful that the gel doesn't float off the glass plate).
  6. Remove the gel from the acetic acid. Cut two pieces of Whatman paper the size of the glass plate and carefully place on top of the gel with glass plate below. Turn the Whatman paper, gel and glass plate over as a unit and then remove the glass plate (the gel sticks to the Whatman). Cover the gel with Saran wrap and dry in a gel dryer for 1-1.5 hours.
  7. Expose film overnight (usually sufficient for 32-P(Potassium isotope 32)) or longer.

Reagents:

Recipes for Acrylamide stocks:

References:

Tabor, S. and C. C.Richardson. (1987) "DNA sequence analysis with a modified bacteriophage T7 DNA polymerase." Proc. Nat. Acad. Sci. USA. 84: 4767-4771.

Sanger, F. Miklen, S. and A. R. Coulson. (1977) "DNA sequencing with chain-terminating inhibitor." Proc. Nat. Acad. Sci. USA 74: 5463-5467.

SequenaseR Step by Step Protocols (1989) United States Biochemical Corporation.