by 50%, thus increasing the average extension length of each primer before a ddNTP is incorporated. Conversely, adding 1∝l of [α-33P]ddNTP will decrease the ratio by 50%, thus decreasing the average extension length of each primer.

Running sequencing gels which resolve more than 600 nucleotides requires high quality apparatus, chemicals and attention to many details. While specific instructions are beyond the scope of this manual, following are some general guidelines: The gel should be loaded with 8 adjacent lanes (GATCGTAC or see ‘Supplementary Information, denaturing gel electrophoresis’ section) with a sharkstooth comb and be run 4 to 10 times longer than usual. For this kind of experiment, gradient (or ‘wedge’) gels or very long gels (80-100cm) are almost a necessity. The highest resolution gels appear to be approximately 6-8% acrylamide and are run relatively cool (40°C).

Denaturing gel electrophoresis

Under optimal gel electrophoresis conditions, 250-300 bases can be read from the bottom of a standard size sequencing gel. The length of time the gel is run will determine the region of sequence that is readable. Many factors can limit the sequence information which can be determined in a single experiment. Among these are the quality of reagents used, the polymerization, the temperature of the gel during electrophoresis, and proper drying of the gel after running. The greatest care should be given to the pouring and running of sequencing gels. The specifics of running the electrophoresis will depend on the apparatus used. The following suggestions for reagent compositions and procedures are intended as guidelines. For specific instructions contact the manufacturer of the gel apparatus used.

Gel electrophoresis reagents

This kit contains a prediluted enzyme mixture which contains a high glycerol concentration, requiring the use of a glycerol tolerant gel buffer. The use of other buffers such as TBE can result in severe distortion of sequencing bands in the upper third of the gel. The following recipe is for typical sequencing gel reagents.

Buffers

20X Glycerol Tolerant Gel Buffer (71949 or 75827)

Tris base

 

 

216g

Taurine

 

 

72g

Na

EDTA.2H

2

O

4g

2

 

 

 

H2O to 1000ml, filter (may be autoclaved)

This buffer can be used with samples containing glycerol at any concentration (20). If gels seem to run a bit slower with this buffer at 1X strength, use it more

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