Question
In preparation for cloning a gene into a plasmid, you haveobtained a 3kb plasmid vector containing two selectable markergenes. One selectable marker renders E. coliresistant to the antibiotic ampicillin. The other selectablemarker, which also contains the multiple cloning site, rendersE. coli resistant to the antibiotic tetracycline. Ifyou successfully clone a foreign piece of DNA into this plasmid atthe multiple cloning site and transform E. coli cells withthe recombinant plasmid, indicate the answer to the followingquestions.
a. How would you select against E. coli cells that havenot been transformed with a plasmid?
b. Which antibiotic(s), if any, will cells be resistant to ifthey have obtained a plasmid that does not contain foreign DNA?
c. Which antibiotic(s), if any, will cells be resistant to ifthey have obtained a plasmid containing inserted foreign DNA?
d. How would you distinguish recombinant and non-recombinantplasmids by agarose gel electrophoresis.
Explanation / Answer
a. How would you select against
E. coli cells thathave not been transformed with a plasmid? Answer: plate them on media containing ampicillin. if thecells transformed, they would be resistant to amp. b. Which antibiotic(s), if any, will cells be resistant to ifthey have obtained a plasmid that does not contain foreignDNA? if they obtained the plasmid in its original form andnone of the DNA you were trying to insert into the plasmid,they would be resistant to both ampicilliin and tetracycline....because those are the two antibiotic resistance genes on thepalmsid. c. Which antibiotic(s), if any, will cells be resistant to ifthey have obtained a plasmid containing inserted foreign DNA? ONLY AMPLICILLIN, if you successfully inserted, then thecloning site is within tet resistance gene, the tet resistance genewon't work anymore. so, the cloned plasmid yields resistance to amponly. d. How would you distinguish recombinant and non-recombinantplasmids by agarose gel electrophoresis. depending on the restriction enzyme(s). choose restrictionenzyme(s) that will cut within the inserted DNA fragment as well asright on the outskirts of it and then look for such band.