I insert a gen to a vector. I must insert a synthetic sequence at this gen by blunt end cloning. At the first stage of cloning (insertion a gen to a vector) is removed negative selection marker against empty vector (ccdB). At second stage when i insert a synthetic sequence at first cloned gen, on LB plate grows clones containing inserted vector (create from second cloning) and none inserted vector (created from first cloning) because negative selection marker not exist in both vectors. How can i know that synthetic sequence insert to gen at second stage of cloning?
You can screen several clones by miniprep and sequencing with internal primers covering your region of interest (or the complete gene). Another thing that you could do is a colony lift. This is a probe based method that allows for screening numerous (all those contained in your plate) bacterial colonies for the desired insert DNA without the need to isolate colonies and analyse separately. You only need to generate a probe matching (specific) your synthetic sequence. In the links provided you will find an example of this protocol. Best of lucks.
http://www.gene.mie-u.ac.jp/Protocol/M&M/Colony-lifts.html
http://www.nhm.ac.uk/resources-rx/files/colony-lifts_aug12-118481.pdf
If you are lucky you can isolate plasmid DNA and cut it with a restriction enzyme that cuts only inside the sequence you added. Linear vector is what you are looking for!
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