topo ta cloning
dk at no.email.thankstospam.net
Wed Dec 29 21:42:18 EST 2004
On Wed, 29 Dec 2004 20:17:09 +0000, ChenHA <hzhen at freeuk.com> wrote:
>> On Sun, 26 Dec 2004 22:46:55 +0100, "as" <nospam at spam.dk> wrote:
>> >Hi. I have a few basic questions regarding topo TA cloning
>> >1) When I TA clone a taq produced PCR fragment into a given TA topo vector
>> >and subsequently sequence or colony screen the clones I usually find that
>> >most of the clones are in the same direction? But there should be random
>> >orientation of the fragments resulting in a 50/50 distribution?
>The reason is usually toxicity of the gene product.
I imagine that could be the case but it's not the explanation I like. The
plasmids we are talking about are straight cloning vectors with no promoters,
ribosome binding site, etc. The toxicity would have to be extreme - which is not
the case of the same genes expressed later in pETs.
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