SUPPLEMENTARY INFORMATION

General guidelines

•Since the popular multiple cloning sites all derive from similar sequences, one primer can serve for the sequencing of insert DNA in most of the common vectors. Among the vectors compatible with the primer supplied in the Thermo Sequenase radiolabeled terminator cycle sequencing kit are M13mp8, M13mp9, M13mp12, M13mp13, M13mp18, M13mp19, mWB2348,

mWB3295, mWB3225, pUC18, pUC19, and virtually any vector featuring blue/white screening with β-galactosidase activity.

•Good sequences can be obtained using as little as 0.05∝g of M13 DNA,

0.1∝g of plasmid DNA, or 50fmol of PCR product. Mix reagents by gently ‘pumping’ the pipettor. The total volume of the reaction mix should be 20∝l— the volumes of DNA and primer added will depend on their concentration.

Adjust the amount of distilled water so that the total volume of DNA, primer and water is 16∝l.

•The specific cycling parameters used will depend on the primer sequence and the amount and purity of the template DNA. See ‘Supplementary Information, cycle conditions and template quantity’.

•The dGTP Nucleotide Master Mix should be used if the sequence is already known to be free of compression artifacts and the benefits of uniform band intensities are desired. The uniform band intensities can aid in finding heterozygotes or in other cases where mixed sequence may be present. If compressions are a problem when using dGTP, gels containing formamide can be used as described in the ‘Supplementary Information, denaturing gel electrophoresis’ section of this booklet.

•For running sequences where compressions are a problem, the dITP Nucleotide Master Mix included in this kit can be substituted for the dGTP Nucleotide Master Mix. See ‘Supplementary Information, elimination of

compressions’ section for details. Note: When using dITP, use an ‘extension’ temperature of 60°C with a duration of at least 4 minutes.

•Whenever possible, tubes should be kept capped and on ice to minimize evaporation of the small volumes employed. Additions should be made with disposable-tip micropipettes and care should be taken not to contaminate stock solutions. The solutions must be thoroughly mixed after each addition, typically by ‘pumping’ the solution two or three times with a micropipette, avoiding the creation of air bubbles. At any stage where the possibility exists for some solution to cling to the walls of the tubes, the tubes should be centrifuged. With care and experience these reactions can be set-up in 15-20 minutes.

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