Blunt end cloning

mnr mnr via methods%40net.bio.net (by mnr475 from gmail.com)
Wed Jul 6 12:23:33 EST 2011


Hi all.

I have tried cloning a potential toxic gene about 1.2 kbp into E.coli with
T7 promoter. After two months, I am still not successful. I am now plannig
to do cloning into pUC18 vector before subcloning into few other vectors
with different promoters. I have limited available restriction sites so that
I don't have to redesign PCR primers. Is it feasible to do blunt-end cloning
with Phusion polymerase into non-dephosphorylated vectors? Will I only be
getting empty vectors after transformations?

Thank you.

Matt


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