Hi everyone. I've been attempting to isolate recombinant pUC 19 from several different
E.coli clones using the NaOH / SDS method. The clones were provided from another student
who had created a library last year from a watewater strain. Here's the problem:
The first time I had run agarose gels, there were no plasmids. Subsequent experiments
had demonstrated that my method could still free up RNA and globs of DNA. I had run a
standard pUC in one lane for the purpose of comparing molecular weights but no other
isolates had shown bands in that region. I have come up with only four answers:
(1) The clones do not harbour any plasmids
(2) The copy number of the existing plasmids is too low to quantify on a gel
(3) The plasmids in question are hybridizing with chromosomal DNA
(4) I am an incompetant with regards to plasmid isolation
Any suggestions would certainly be appreciated. I'm about to consider
transforming a strain myself to ensure that the vector is in the cells instead of going
on a student's word.
Yours truly, Neil M.
