Help for site-directed mutagenesis

[fei wang]

Hi netters:



IÕve just started to perform site-directed mutagenesis. Could 

I use the pBluescript(pBS) phagemid and CJ236 bacterial 

strain with VCSM13 helper phage to replicate my single-

stranded(ss) pBS DNA? Second question is someone only gives 

me a ssDNA pBS phagemid (inserted gene) with dUTP. Could I 

replicate this DNA in CJ236 bacteria without uridine and then 

replicate it in DH5-alpha bacteria to get my double-

stranded(ds) DNA without dUTP? If it can work, is there any 

possible to have mutation sites in the pBS phagmid in this 

procedure? Third question is someone gives the protocols of 

manipulate the ssDNA with VCSM13 helper phage, but this 

protocol does not add any kanamycin in culture process. Does 

it work to let me to get ssDNA of pBS phagmid?



I appreciate any advice on this matter.



Yours sincerely,

Tseng-yuan Chang

1995/11/30/14/44min