PCR product sequencing

Cameron McCrae (cameron.mccrae@anu.edu.au)
Fri, 18 Jun 1999 09:56:42 +1000 (EST)

In response to the recent messages regarding sequencing of PCR products...

Large scale sequencing of PCR products is a regular event in our lab (Human
Genetics, John Curtin School of Medical Research, Australian National
University). We have no trouble with it at all and I have never needed to
clone PCR products to enable sequencing.

A strategy that my lead to better sequence is to do a nested PCR (ie. do
the PCR and use the product of this as the template for a second PCR that
uses an internal primer pair, and then sequence this product). Nested PCRs
can help eliminate nonspecific products. It may help to purify the PCR
product before sequencing it (to remove unused primer and dNTPS; I have
used both Millipore and Qiagen kits for this and they are available in 96
well format).

I you want anymore info please contact me <Andrea.Mettenmeyer@anu.edu.au>.