plasmid isolation problem HELP! - (Dec/01/2008 )
I'm having problems isolating my plasmid in miniprep also maxiprep. My plasmid is pML7 (pmon17336) and i'm using E.coli glg C (-) for transformation.As i got colonies after my cloned product transformed E.coli glg C (-) (i also tried DH5 alfa) i confirmed my insert by colony PCR(Lysate PCR). But when i tried to extract Plasmid by Alkali Lysis method i get plasmid in very high band after elctrophoresis. i tried to double digest it 1ul didn't work but 10ul worked.then i tried maxi prep i didn't get any thing.what must i do? please someone help.
by the way my plasmid resisted to spectinomycine /streptomycine.i'm using strep.
It sounds like your extraction wasn't very good: lots of genomic DNA. Be careful with the transfer of supernatants over to the fresh tubes, also be careful with the spin steps.
And never forget to grow the coli cells with antibiotics