methods to check orientation and frame in pQE30 - (Jun/27/2006 )
I would like to know how one can determine the orientation of a gene cloned in pQE30 in detail and if is it possible to to identifiy in-frame genes in a clone of BamHI (double digest)from some 100 transformants as there is no blue white selection in this expresion vector.
thanks for any reply
I'm new to this and any reply would b of great help
Iam using pQE30 for expressing a 26KDa protein and Iwould like to know the possible methods to screen the recombinants in frame before going for any purification and induction.I've got nearly 100 transformants from which i 've to screen the ones expressing the protein . Please help me out
Avery needy jmj
don't know pQE30 (which company) - does it have blue/white selection?
with this you can select plasmid containing insert from empty plasmid but to be sure that the insert is really in frame (of course you should have designed and cut insert properly) you will have to sequence...
what do you think about sequencing your expression clones. pQE is a standard vector with T7 promotor. So sequence it with T7 forward primer.
digest BamH I and an other enzyme in your insert. The mix should give a different pattern in the 2 situations (sense and antisense).
For checking in frame, you'll need to sequence the relevant clones mapped in exp 1.
You should search for a RE that cuts in the 5' or 3' of your cDNA and it doesn't cut or cuts only once in your vector. If it doesn't cut in the vector, digest also with another RE that cuts once upstream or downstream your insert, so that you can see 2 bands: both of them are vector+ a part of the insert. You should be able to discriminate how your cDNA was inserted in your vector in this way
To determine orientation, you could colony PCR using a vector primer and an insert primer, where the orientation controlled whether amplification occurs or not. Why not simply sequence, which will tell you more, including identity and frame.