Is there a sureshot method for double-stranded plasmid sequencing?
This answer comes from Sasha Kraev (bckraev@aeolus.ethz.ch). This question may be reduced to “How can one prepare a good template from plasmid for polymerase `Sanger sequencing’?”. The original paper of Chen.Seeburg1985 described the use of CsCl centrifugation and alkali denaturation that produced DNA ready to be used in the original protocol from Sanger1977 with the Klenow fragment of DNA polymerase I. New DNA polymerases, particularly phage T7 DNA polymerase, has allowed to use miniprep DNA for sequencing in a reliable way. The most widely used method utilizes a T7 polymerase/Sequenase kit and an alkali denatured miniprep. However, the quality of DNA, produced by any simple miniprep, is subject to variations, the most important of which are particular clone yield, strain used and individual skills. Since any miniprep procedure results in a defined amount of contaminants in the plasmid DNA, clones with low yield are generally concentrated in a small volume; this increases the chances
