Subject: amplification of DNA fragments >5kb

From: [email-masked] (Barbara Howlett)
Date: 15 Aug 1996 22:47:18 -0700

We are attempting to amplify the non-transcribed intergenic spacer of 
ribosomal DNA of the ascomycete Leptosphaeria maculans.  Southern 
analysis indicates that there are different sized rDNA repeat units in 
individual L.maculans isolates, and we expect that this variation is 
within the intergenic spacer, which probably contains different 
numbers of repeated elements. 

In experiments using Promega Taq or Tth DNA polymerase we have been 
unable to consistently amplify fragments longer than 3.5 kb, although 
we expect some intergenic spacers to be up to 5.5 kb. Does anyone have 
any experience with PCR systems that can amplify fragments of over 5 
kb which contain repetitive sequences?

Thanks
Barbara Howlett
School of Botany
University of Melbourne
Parkville 3052 VIC
Australia
phone 613 9344 5062
FAX   613 9347 1071