(by k.vd.wetering from nki.nl)
Wed Jan 23 12:16:24 EST 2008
no I am not using BP clonase to clone our gene of interest into the
pEntr vector. Instead, I want to subclone it from another non Gateway
construct that contains my gene of interest.
Felipe da Veiga Leprevost wrote:
> Dear all,
> " I want to use the gateway system of invitrogen to minimize the
> workload of subcloning our cDNAs in various expression vectors.
> using the system I have to clone our cDNAs in an appropriate pEntr
> vector. I used pEntr1a for this but I do not get any colonies on
> kanamycin plates using homemade DH5alpha or Invitrogen Top10 cells.
> did remove the ccdB sequence before putting in our cDNA. I also
> cutting out the ccdB sequence, religating the empty vector and
> transforming bacteria with this. I get only a very limited amount of
> colonies from transformed Top10 cells and none on our own competent
> Hi Koen,
> What protocol are you following to enter the plataform? Are you
> making the BP reaction to clone your DNA into the entry vector?
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