Plasmid DNA

The plasmid DNA should be of high purity with no contaminating RNA, salt, ethanol, EDTA, protein, or Phenol. Read lengths for high quality and pure plasmid DNA can be out to 1000 bases.

Recommended Purification Procedures and/or Kits:

• Qiagen Plasmid Purification Kits Qiagen offers various plasmid purification kits depending on your throughput needs
• Ultracentrifugation in a CsCl density gradient Care must be taken to remove residual CsCl from the DNA either by dialysis and/or ethanol precipitation.
• Promega Membrane based Kits

Important Steps:

• Wash the DNA pellet at least once with 70% ethanol. Ethanol removes any residual salt from the DNA. Salt will interfere with Taq polymerase activity resulting in low quality sequence data.
• Dry the DNA for 15 minutes in a speedvac to remove residual ethanol after the ethanol wash step. Ethanol will cause a dramatic reduction in signal strength, resulting in noisy sequence.
• Use the recommended quantity of LB broth for cell growth. Overloading the Qiagen resin can result in poor yield of plasmid DNA, presumably due to competition with RNA fragments for binding.

PCR Products

Amplified DNA submitted for sequencing must be free of PCR primers, RNA, salt, EDTA, protein and ethanol. Any residual PCR primers will act as sequencing primers and therefore produce noisy sequence.

Recommended Purification Procedures and/or Kits:

• Qiagen QIAquick If after using the kit your sample shows evidence of PCR primer contamination, performing the wash step 3 times has been shown to resolve the contamination issue.
• Preparative agarose gel electrophoresis
• Sephadex chromatography
• Shrimp alkaline phosphatase with Exonuclease I Effectively removes primers