|Register||Search||Today's Posts||Mark Forums Read|
|PCR - Polymerase Chain Reaction Forum PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.|
| ||LinkBack||Thread Tools||Display Modes|
qPCR of GC rich region
I have a serious trouble here: I am trying to qPCR a ~300bp region with 72% GC content without any luck so far. I was running a load of optimizing reactions: including temperature gradient for anealing with addition of DMSO 0; 2 and 5% . I am using the Brilliant II SYBRŪ Green QPCR Master Mix from Stratagene with Hot Start Polymerase (therefore initial denaturation step was 10min at 95C), Mix has 2.5mM MgCl2, which is a relatively high concentration. The only thing I haven't tried yet is the TD-PCR, but it doesn't work for quantitative purpose...
Any ideas, suggestions to continue with? Perhaps raising the denaturation temperature/time? The melting temperature of region is higher than 100C, but won't it kill polymerase even though it is a hot start enzyme?
Could it also be a primer issue: one of the primers has a Tm around 62, the other one around 72 (and 73% GC content)?
Any other thoughts???
The only thing I know for sure: it shouldn't be a template issue, another target works perfectly.
Thanks in advance,
|qpcr , region , rich|
|Thread||Thread Starter||Forum||Replies||Last Post|
|qPCR NEWS January 2009 - update in available real-time PCR cycler||Editor www.Gene-Quantification.info||Protocols and Methods Forum||0||02-19-2009 10:04 AM|
|qPCR Newsletter July 2008 - main focus on qPCR optimisation||Editor www.Gene-Quantification.info||Protocols and Methods Forum||0||07-28-2008 02:48 PM|
|qPCR NEWSLETTER - September 2007 - qPCR 2007 talks online||Editor www.Gene-Quantification.info||Protocols and Methods Forum||0||09-25-2007 12:34 PM|
|qPCR NEWSLETTER - Final call for qPCR 2007 Event||Editor www.Gene-Quantification.info||Protocols and Methods Forum||0||02-28-2007 02:01 PM|
|qPCR 2007 - CALL for scientific contributions||Editor www.Gene-Quantification.info||Protocols and Methods Forum||0||01-22-2007 01:24 PM|