PCR product size with a Roche Lightcycler? - (Jul/08/2005 )
Wasn't sure which forum this would really fit into but here goes. I am working on methylation studies right now and would like to amplify an entire CpG island(3 islands actually with product sizes ranging from 500-1000 bp).
Has anyone had any experience with amplifying such large products? What types of problems would I be likely to experience if I were to try? If worse comes to worse I could break the islands up into smaller amplicons, but I would rather do it in one shot if possible. Typically in the lab we work with 200-300 bp products amplified with the Lightcycler and qiagens one-step RT PCR kit.
-CW
-cwong1215-
QUOTE (cwong1215 @ Jul 8 2005, 04:47 PM)
Wasn't sure which forum this would really fit into but here goes. I am working on methylation studies right now and would like to amplify an entire CpG island(3 islands actually with product sizes ranging from 500-1000 bp).
Has anyone had any experience with amplifying such large products? What types of problems would I be likely to experience if I were to try? If worse comes to worse I could break the islands up into smaller amplicons, but I would rather do it in one shot if possible. Typically in the lab we work with 200-300 bp products amplified with the Lightcycler and qiagens one-step RT PCR kit.
-CW
Has anyone had any experience with amplifying such large products? What types of problems would I be likely to experience if I were to try? If worse comes to worse I could break the islands up into smaller amplicons, but I would rather do it in one shot if possible. Typically in the lab we work with 200-300 bp products amplified with the Lightcycler and qiagens one-step RT PCR kit.
-CW
The Lightcycler can amplified products up to 1 kb. I have amplified large product but nothing above 600 bp.
Key to optimization, treat the lightcycler like a regular thermalcycler. A one second denaturation isn't going to work on larger size amplicons. A 15-45 sec denaturation, 30-1min sec-anneal and 1-3 min extension will probably be a good starting point.
You're going to need a greater concentration of Taq, MgCl and dNTPs.
-dobbiewalton-