Plasmid gene mutation - (Oct/23/2004 )
I have some Tn10 derivative trasposon in plasmid vehicle cloned in E.coli. Example of them are - Derivative 105,marker in transposon is CamR and venicle is plasmid pnK2284,and deirivative 108 ,Markers in transposon is CAmr ,Plac ,plasmid vehicle is pNk2887.
I want use them for random mutagenesis in Pseudomonas.The gene of my intrest is in Plasmid.
Can you please suggest the protocol for mutagenesis using these transposon to mutate plasmid gene.
Thanking you in advance
use very cheap taq to amplify the insert off a template plasmid. Taq is error prone and will introduce random mutations into your dna. design primers that have restricion sites in them (or add them yourself) so that you can easily whack your pcr products back into the vector.
Adding extra MgCl2 to your pcr increases the error rate (as does adding dmso).