cloning the wrong way?
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Wed Feb 16 11:26:36 EST 2000
In article <88ee35$b6u$1 at nnrp1.deja.com>, ahmed.elgedaily at dim.usz.ch
>When I pick the tiny colonies that are on the plate and prepare them
>I get the insert in the desired orientation.
>But I cannot grow these clones more than once!
>Do you have an explanation why this happens?
>Is the insert toxic to the bacterial cells?
Looks that way. Presumably the lethal orientation is the same one as
expression of the lac alpha fragment.
>Shall I use a different cloning system?
Pick an insert in the opposite orientation that sequences OK and
subclone from that.
>Do I have to sequence the clones to make sure that they are the right
>DNA and to confirm the orientation?
Verify the correct insert by PCR or restriction map then sequence. The
only way you will clone it in the opposite direction is to put it in a
vector such that there is no promoter in the direction you want to clone
it or that the promoter is totally repressed. You may be able to get
your existing small clones to grow in the presence of glucose i.e.
better repression of the lac promoter.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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