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Introduction

• A typical sequencing will produce 700 bases of sequence. Many runs will yield 1000+ bases. As with all procedures garbage in, means garbage out. Poor quality / dirty samples, poor or degraded primers or too little or excessive DNA and / or primers will result in poor or failed sequence.

• Use your preferred method to isolate plasmid DNA. Need help?  See the MICB Sequencing Guide.

• Too little template is bad as is too much. Try to provide the amount of DNA indicated in the Sequencing Procedure.  

• 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 or the cheaper and faster MiniClean Kit (2MCL-05) from Gel COMPANY. 

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