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Introduction

  • A typical sequencing will produce 400 bases of sequence. Many runs will yield as much as 500 bases. As with all procedures garbage in, means garbage out. Poor quality / dirty samples, poor or degraded primers or excessive DNA and / or primers will result in poor or failed sequence.

  • Use your preferred method to isolate plasmid DNA. Need help? Try Don's miniprep method. We have observed consistently better sequencing results where the DNA samples have been phenol / chloroform extracted. This is true even with CsCl purified DNA. Our phenol extraction procedure is described below. DO THE EXTRACTION CAREFULLY SINCE  TRACES OF PHENOL OR CHLOROFORM WILL INHIBIT THE SEQUENCING REACTION.

  • Too much sample yields poorer results. Try to provide the amount of DNA indicated in the Sequencing Procedure. Remember, more IS NOT better.

  • Where possible use a primer selection program to select high stringency primers for your sequencing. Where a poor primer must be used, try adding up to 4x more primer.

  • Dissolve your sample DNA in ddH2O and not TE as EDTA inhibits the sequencing reaction.

  • Always use 100% ethanol for precipitations. Impurities in 95% ethanol will inhibit the sequencing reaction.

  • If you suspect poor quality DNA samples, ABI recommends Qiagen purification kits like the Qiagen QIAprep Spin Miniprep Kit (50) Cat.#27104 for plasmid minipreps. We have also successfully used Sigma's GenElute Plasmid Purification MiniPrep Kit, Product Code PLN-10 (10) or PLN-70 (70 purifications).

  • If you are using the Qiagen QIAprep Spin Miniprep Kit ALWAYS BOIL the isolated DNA prior to quantitation. We have noticed that Qiagen Miniprep DNA is sometimes somewhat "insoluble" when isolated and that boiling the sample resolves this problem.

  • If you are having problems sequencing PCR products use the ExoSAP-IT kit (US78200) from Amersham Pharmacia Biotech [MICB Biobar] for rapid and efficient purification of PCR products. To 5ul of your PCR product add 1ul ExonucleaseI and 1ul Shrimp Alkaline Phosphatase. Incubate at 37C for 15 min and then 80C for 15 min. You would then use 0.5-2ul of this purified PCR product for sequencing.

Standard Phenol Extraction (Precaution: Phenol is very corrosive....Handle with care and read the MSDS if in doubt)

  1. To your DNA sample add an equal volume of buffer saturated phenol:chloroform: isoamyl alcohol (25:24:1)[Sigma-Aldrich, Cat# P-3803] (be sure that your stock phenol is at neutral pH). Vortex, microcentrifuge for 5 min (>12K rpm) and remove the top aqueous layer to a fresh eppendorf tube.

  2. To remove phenol, extract again with an equal volume of chloroform:isoamyl alcohol (24:1)[Sigma-Aldrich, Cat# C-0549]. Vortex, microcentrifuge for 2 min and remove the top aqueous layer to a fresh eppendorf tube.

  3. Precipitate the DNA by adding 1/15th volume 3M sodium acetate pH5.2 and 2.2 volumes 100% ethanol.

  4. Precipitate at -70°C for 15 min or at -20°C overnight.

  5. Pellet DNA by microcentrifugation for 15 min. Remove all ethanol and dry the DNA pellet. Redissolve the DNA in an appropriate volume of ddH2O. Quantitate.