HomeBrew, on 08 September 2010 - 03:15 AM, said:
Shaking of the culture is done to insure adequate aeration. A overnight static culture of E. coli should still yield plenty of cells for a miniprep -- you should have seen plasmid bands with or without restriction enzyme treatment. You may be confusing sequencing with colony PCR -- you don't need to isolate plasmid DNA to see if your plasmid + insert is in any given colony -- you can do that by PCR with appropriate primers right from the colony. You do, however, need to isolate plasmid DNA to sequence the plasmid or insert.
I see... Thanks for the information!
adrian kohsf, on 08 September 2010 - 03:25 AM, said:
Did you include proper antibiotics when you incubate your E.coli? Yes, I agree with Homebrew, you can actually do a colony PCR on it to verify whether the insert is inside your E.coli before you proceed for miniprep.
Usually for an established protocol, a miniprep will be hardly goes wrong.
I suspect you had lost your recombinant strain and the clump might be something else.
Just my 2 cents.
Yes i did include antibiotics with the same concentration as my plate.
Do you mean that the recombinant strain is lost when transferring from plate to broth? OR it lost its function? What can i do for that?
I ran 2ul sample in gel. Tomorrow will try 5ul. Hopefully its because of low concentration..