gateway system

| |Koen via methods%40net.bio.net (by k.vd.wetering from nki.nl)
Tue Jan 22 11:06:06 EST 2008


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.

can anyone tell me what I did wrong?

Koen


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