Gateway System

[Felipe da Veiga Leprevost]

   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