I need to know how to design GSPs for RACE. Iam going to use Clontech Smarter RACE kit for the first time. My RNA quality is good. Even the GSPs I designed for semi-quantitative works well. Whether I should use the same GSPs or design other ones with higher Tm. My question is how to proceed with cloning my gene using RACE amplification, starting with the primer design for the same.
Iam sending the link of the related paper. http://www.sciencedirect.com/science/article/pii/S0161589009007664
I am also using RACE Strategy and we have successfully cloned our 3'end ,
for you : if you know partial fragment and you don't know the 3' or 5' end you could go for 3' or 5' RACE. what you need is your partial sequence and 3 primers which binds Internal to each ....
I am also using RACE Strategy and we have successfully cloned our 3'end ,
for you : if you know partial fragment and you don't know the 3' or 5' end you could go for 3' or 5' RACE. what you need is your partial sequence and 3 primers which binds Internal to each ....
Thanks Prakash.......I have a primer for my gene of interest whose product size is 147bp and it works well in semi-quantitative PCR. Can I use that primer for RACE or design new one? Well, Clontech Smarter RACE kit clearly mentions to use GSPs with higher Ta. So like what suggestion do you have for this
You could use that primer ...but only one primer is not sufficient na. You require 3 primers which are internal to each other......
[email protected] is my email address. Thanks alot Sir, for replying. I was really in need of the paper. And Sir, if I have any queries regarding the paper would you mind if I mail you?
The kit comes with instructions on how your GSP primers should be. It is online and it is different that normal primers. The Tm is much high 60 and greater and the primer length can be 23-28. etc.
The primers for 3' RACE are the same sequence while the primers for 5' RACE are the reverse complement
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