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gene cloning - (Apr/07/2013 )

I need to clone gene, whose only one cd sequence is available on NCBI and is 3534 bp long, when i am doing transformation and using DH5a as host I am not getting any clone on substrate plate. Suggestions needed,


What is the plasmid you are using? some of them are not good for such large fragments...

Did you checked whether the fragment is correct after PCR?

Another good thing - run PCR with plasmid primers prior the cloning to make sure, that you have at least some plasmids with an insert. So then you can try to dig up the roots of the problems :)