How to joint two cloning genes?

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How to joint two cloning genes?

Post by rabbitdoctor » Fri Apr 08, 2011 9:39 pm

I want to construct a fusion gene, which comes from chromosome translocation.
I have the sequences of the two DNA fragment. But how can I joint them together?
One ends AACAGG
One begins TCCTCC
I input the sequence AACAGGTCCTCC, and try to find a restriction enzyme to cut them.
But they gave me four enzymes: AvaII, EcoO109I, PpuMI, Sau96I.
The sequence of Sau96I is GGNCC. What does N mean?
Can I use this enzyme to joint two genes?
Or is there any other way to joint the two genes?
I don't want to add enzyme site between two genes, for that maybe interfere the fusion gene's function.

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Post by JackBean » Sat Apr 09, 2011 1:37 pm

and why do you need to add restriction place? You could use simply fusion PCR

Cis or trans? That's what matters.

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Re: How to joint two cloning genes?

Post by domwood » Tue Apr 12, 2011 3:13 pm

Blunt Ended Ligation?

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Post by BodhiBio » Mon May 23, 2011 9:55 am

You could try Slicing by Overlapping PCR (SOE-ing), its very easy. You just need one overlapping primer :)

Source: Karin L Heckman & Larry R Pease. Gene splicing and mutagenesis by PCR-driven overlap extension. Nature Protocols 2, - 924 - 932 (2007)

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