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.