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About 5' Race

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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About 5' Race

Postby boyzoe » Wed Dec 09, 2009 9:00 am

Genome of my research species haven't sequenced yet. So after blast with other species, I designed primers and sequenced a fragment for the gene I researched in the species. Then I used 3' Race cloned the 3' fragment. However, I don't know how to get the sequence of 5'.
I know that Smart Race of Clontech can solve the problem. Is there other way to obtain 5' sequence of cDNA?
I propose no commercial kit will be used.
And if you guys have any experiences about 5', I hope you can talk of your method.
Thanks!
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Postby JackBean » Fri Dec 11, 2009 8:11 am

What species are you working with?

What NA do you use? cDNA? I would suggest to do gDNA library, there you can perform 5' RACE, can't you?
http://www.biolib.cz/en/main/

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Re:

Postby boyzoe » Sat Dec 12, 2009 3:01 am

JackBean wrote:What species are you working with?

What NA do you use? cDNA? I would suggest to do gDNA library, there you can perform 5' RACE, can't you?


The species is goldfish.

We haven't created gDNA yet. I cloned the gene from cDNA and sequenced 3' fragment. Blasted with zebrafish, there are almost 400bp to ATG. I want to obtain the full length of the gene.

Really grateful for your reply!
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Postby JackBean » Sat Dec 12, 2009 3:31 am

you mean, that you miss 400bp of 5' part of gene?
Anyway, I think, even with cDNA this could work. Just design primer like in middle of gene and do the sequencing, you should get at least something ;)
But otherwise, gDNA is the best choice in my opinion ;)
http://www.biolib.cz/en/main/

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