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primer design tips - (Jul/01/2010 )

Hey everybody,
I'm trying to establish real time PCR in our lab, on a RotorGeneQ. Somehow I can't get my primers to an efficiency higher than 70% or 80% and if it gets close to 90% the y-axis intercept is too low or high or I start having primer-dimers. Currently I am using Primer3 for the design and then check them with OligoClac and OligoAnalyzer for self-hybridization and dimer formation. Annealing is for 15s at 60C and elongation 20s at 72C, the product size is around 150bp.
I am running out of ideas, does anybody have hint how to improve?

-Xisto-

Xisto on Jul 1 2010, 09:40 AM said:

Hey everybody,
I'm trying to establish real time PCR in our lab, on a RotorGeneQ. Somehow I can't get my primers to an efficiency higher than 70% or 80% and if it gets close to 90% the y-axis intercept is too low or high or I start having primer-dimers. Currently I am using Primer3 for the design and then check them with OligoClac and OligoAnalyzer for self-hybridization and dimer formation. Annealing is for 15s at 60C and elongation 20s at 72C, the product size is around 150bp.
I am running out of ideas, does anybody have hint how to improve?



This strikes me as bit of a bizarre issue...not sure what sort of qPCR your doing, but have you tried it with published primer sequences for oft-used targets (e.g, GAPDH for RT-qPCR, or something like LINE1 for qPCR from genomic DNA). I've done quite a bit of primer design for a genetic region that has some tough spots in it, and I haven't run into an issue like what you mentioned. Do you also use PrimerBLAST to check your primers to make sure you're not picking up something else? If not you probably should.

MM

-Mighty Mouse-

This strikes me as bit of a bizarre issue...not sure what sort of qPCR your doing, but have you tried it with published primer sequences for oft-used targets (e.g, GAPDH for RT-qPCR, or something like LINE1 for qPCR from genomic DNA). I've done quite a bit of primer design for a genetic region that has some tough spots in it, and I haven't run into an issue like what you mentioned. Do you also use PrimerBLAST to check your primers to make sure you're not picking up something else? If not you probably should.

MM




Hey!
Thanks for the reply. Well, I'm using EvaGreen and currently I am looking for housekeepers (I'd like to do it as in Vandesompele 2002). The problem is that I have to veryfy data from microarray experiments and the primers other people used for housekeepers I cannot use, cause I find a regulation in the array for the same genes. Plus we are working with a non-model organism. And yes after design I check the primers for unspecific reactions in silico as well as in a PCR, where I also look at the one-primer reactions.
So how did you do your primer-design? Did you use any program or did you do it by hand?
Thanks

-Xisto-

Hey!
Thanks for the reply. Well, I'm using EvaGreen and currently I am looking for housekeepers (I'd like to do it as in Vandesompele 2002). The problem is that I have to veryfy data from microarray experiments and the primers other people used for housekeepers I cannot use, cause I find a regulation in the array for the same genes. Plus we are working with a non-model organism. And yes after design I check the primers for unspecific reactions in silico as well as in a PCR, where I also look at the one-primer reactions.
So how did you do your primer-design? Did you use any program or did you do it by hand?
Thanks



I did my primer design just like you...primer3...then NetPrimer then PrimerBLAST. The reason I was suggesting the use of housekeepers others have used is simply to demonstrate that you can demonstrate primer efficiency with primers that are supposed to be efficient, that way you can rule out any issues with your general mastermix, or machine and you know it has to do with your primer design. For what it's worth I use the sybrGreen master mix from ABI.

MM

-Mighty Mouse-