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DNA Sequencing Facility

DNA Sequencing

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Primers

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Template Prep

Sample Sheet

Instructions

Instrumentation

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Template Preparation (from ABI Sequencing Guide)

The most reliable DNA plasmid purification kits are Qiagen, Biorad, and Promega Midi kit. When using a bacterial host for plasmid production, HB101 and DH5a strains consistently produce good results, whereas MV1190 and XL1Blue lack consistency. JM101 generally does not work well.Recommended PCR cleanup methods include Qiagen and Wizard kits, Microcon concentrators, and USB PCR pre-sequencing kit 70995 or 70996. The following list describes the types of templates that are most difficult to sequence:

• G-C content > 70%
  (courtesy of Dr. Richard Eberle, Oklahoma State University)
• an overall GC rich base composition
  (courtesy of Dr. Richard Eberle, Oklahoma State University)
• an overall AT rich base composition
  (courtesy of Dr. Chris West, OUHSC Department of Biochemistry and Molecular Biology)
• extremely AT or GC rich regions
• regions of pronounced secondary structure
  (courtesy of Dr. Myron Hinsdale, OMRF Department of Cardiovascular Biology)
• long triplet repeats regions
  (courtesy of Dr. Sanjay Bidi3handani)
• containing long repeats