|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|
PCR Protocol Questions
Im designing four different PCR protocol/program for the first time for a cloning project and I could use some help. My issue is with the melting temperatures for the oligos I have; some of them are very high. For one reaction, I have a 78.2* and 92.3* Tm for my primers and another reaction has 92.2*, 88.3* and 78.2* (I am combining sequences in this rxn)
I dont know how to choose a correct annealing temperature for these two reactions. I know the standard way is denature-1' @94*::anneal- 1' @Tm-5*::Extension 3' @72*.
Are these oligos with the really high Tm useful? How can I go about choosing a good annealing temperature? I have only a few weeks left in this project so I cannot start over
Please help! I would love any advice you can give and will answer any questions that I can.
P.S. I should note that the two oligos in the 90s have tails that I am using to attach a new shortish sequence.
Last edited by wanderingstranger; 02-21-2011 at 08:57 PM.
|annealing temperature , melting temperature , pcr , protocol , questions|
|Thread||Thread Starter||Forum||Replies||Last Post|
|100 Questions for Plant Science Research||Claire Grierson,School Biological Sciences||Arabidopsis and Plant Biology||2||02-11-2013 07:00 AM|
|Matrigel Invasion Assay Protocol||moleculardude||Cell Biology and Cell Culture||6||06-11-2012 12:38 PM|
|100 Questions Deadline JUST TWO WEEKS AWAY||Claire Grierson,School Biological Sciences||Arabidopsis and Plant Biology||0||03-17-2009 03:52 PM|
|Protocol for human CD38 WB||Morales, Victor||Protein Forum||0||02-02-2008 08:30 PM|
|Protocol: Inner and Outre membrane Isolation?!||Natalia Oliveira||Protocols and Methods Forum||1||10-09-2007 10:31 PM|