PLASMIDS

Plasmid DNA:

Purity

The DNA should be of high purity with no contaminating RNA, salt, ethanol, EDTA, protein, or Phenol.

            Recommended Purification Methods: Qiagen offers several quality kits that vary in scale and throughput.   Please refer to Qiagen Sequence Guide to find the kit that is most appropriate for you: http://www.qiagen.com/literature/brochures/seqguide/seqchoos.asp    

Although the Qiagen kits are very reliable, there are some common mistakes which compromise the final quality of sequence data:

• Wash the DNA pellet at least once with 70% ethanol.  Residual salt in the final template will interfere with the activity of Taq polymerase.  This often results in “dirty” sequence data with a low signal to noise ratio.  Sequences with excess salt typically run shorter than expected.   
• To remove residual ethanol, dry the DNA for 15 minutes in a properly operating speedvac. Residual ethanol will cause the signal to decrease dramatically, resulting in a noisy sequence.
• Use the recommended quantity of LB broth (don't use Terrificbroth) for cell growth.  Overloading the Qiagen resin can result in a poor yield of plasmid DNA, presumably due to competition with RNA fragments for binding.

In addition to the Qiagen procedure, there are two other recommended methods for consistently preparing high quality template DNA:

• Ultracentrifugation in CsCl density gradients often yields excellent template DNA (these are especially good for BAC DNA).  Following centrifugation, one must carefully remove residual CsCl from the DNA either by dialysis and/or ethanol precipitation (Cs inhibits Taq polymerase).
• Promega purification kits also work well for automated sequencing. Strict adherence to the instructions is necessary when using any plasmid purification kit.

Quantity

Besides the method of plasmid preparation, the quantity of plasmid is an important determinant of the accuracy and reliability of the sequence data. Plasmid DNA concentration should be accurately measured (by UV absorbance, fluorometer, or by ethidium bromide stained agarose gel electrophoresis. Too much or too little DNA are both reasons for a failed sequencing reaction. Too little template results in reactions with little or no signal and poor basecalling. Too much DNA produces reactions that terminate prematurely, often with less than 250 bases of reliable sequence data.

For most plasmids, provide DNA at 100ng/µl (at least 10ul per reaction) in water (preferable) or 10mM TE.