plasmid isolation problem

[Chinnu]

Hi,

I am facing one problem with plasmid isolation. nw i am doing one project wrk on pcr mutagnesis.

transformation of the pcr product gave one colony after 12 hrs. i isolated the dna from the colony( XL-1 blue supercompetent cells were used for transformation)
But the concentration is very less, i.e; appro.. 25ng/ul

so i again transform the isolated plasmid to DH5 alpha cells to get more.

But it also gives lowest concention.I have done these things repeatedly.

I am using the phenol chloroform method for the plasmid isolation.According to the competent cells used,is there any change in the protocol..

please help me, if i didn't get enough plasmid, i am not able to send it for sequencing.


please.......................