What methods can be used to verify that transformation occurred? Besides using ampicillin resistance, what methods can be used to verify that transformation occurred?
kwicher replied: "If you want to know in general what selection method one can use:
- resistance to other antibiotics (chloramphenicol, kanamycin, zeocin )
- use of auxotrophic strains, which are unable to grow in the absence of some particular compound and plasmids which provide gene allowing for synthesis of this compound
(http://en.wikipedia.org/wiki/Auxotrophic)"
archaeadoc replied: "Well, transformation requires the transfer of something. So far you have talked about the thing you can see, amp resistance. That would be the ability to grow on a plate with amp. but what is being transfered? If it is a plasmid, then you could look for the plasmid (plasmid mini prep, digest, run the gel). If it is some other genetic material you could look for the presence of that genetic material (genomic DNA prep, digest, southern transfer and probe for the new stuff). You don't give enough info here to know what is being transformed, but in the case of a plasmid, amp resistance is not enough. You can from time to time find spontaneous mutants that will grow on amp (not many, but it's happened to me). In those cases they can grow a little on the plates, but don't do well at all in liquid."
Is it possible to use kanamycin antibiotic instead of ampicillin for LB agar plate for gene expression? I am planning to grow the colony from a host plasmid in LB agar plate. I used Kanamycin instead of ampicillin but I didn't get any colony after 16 hour incubating at 37degree centigrade temperature.
What can be the possible mistake that I might have made?
Any suggestion would be appreciated
dooberheim replied: "The plasmid that contains the gene you want to express confers resistance to penicillins (beta-lactamase). Kanamycin is an aminoglycoside, and requires a different plasmid (one that confers kanamycin resistance. The kanamycin killed your colonies. Make new plates with ampicillin and it should work.
DK"
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