Gateway System

[| |Koen]

Hi Felipe,

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.

Koen

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.
>    Before
>    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.
>    I
>    did remove the ccdB sequence before putting in our cDNA.  I also
>    tested
>    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
>    DH5alpha."
>
>    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?
>    Leprevost