You want to clone gene A (1000 bp) into the SalI restriction site of the vector pBR322 illustrated below. pBR322 contains both ampicillin and tetracycline resistance genes. a) Which antibiotics would you include in the agar plates that would allow bacterial cells to grow that have been transformed with: i) No plasmid. ii) Non-recombinant pBR322. iii) Recombinant pBR322 with gene A cloned into the SalI site. b) Assume the cloning of gene A was successful, and you have isolated the recombinant plasmid. You set up separate restriction enzyme digestion for the non-recombinant and recombinant plasmids with: (i) SalI (ii) PstI + NdeI. Draw the gel profile you expect to observe when you subject the products of these four reactions to agarose gel electrophoresis. Indicate the sizes of all bands on the gel.
Added by Felix T.
Step 1
For bacterial cells transformed with non-recombinant pBR322, include both ampicillin and tetracycline in the agar plates. For bacterial cells transformed with recombinant pBR322 with gene A cloned into the SalI site, include ampicillin in the agar plates. ** Show more…
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You want to clone gene A (1000 bp) into the SalI restriction site of the vector pBR322 illustrated below, pBR322 contains both ampicillin and tetracycline resistance genes. a) Which antibiotics would you include into the agar plates that would allow bacterial cells to grow that have been transformed with: i) No plasmid. ii) Non-recombinant pBR322. iii) Recombinant pBR322 with gene A cloned into the SalI site. b) Assume the cloning of gene A was successful, and you have isolated the recombinant plasmid. You set up separate restriction enzymes digestion for the non-recombinant and recombinant plasmids with (i) SalI and (ii) PstI + NdeI. Draw the gel profile you expect to observe when you subject the products of these four reactions to agarose gel electrophoresis. Indicate the sizes of all bands on the gel.
Shaiju T.
Assume you have a plasmid with an origin of replication, a selectable marker (strR) which when functional confers resistance to the antibiotic streptomycin and a multiple cloning site (MCS) within the coding region of the ampicillin resistance (ampR) gene which when functional confers resistance to ampicillin. (note: s = sensitive) Under normal circumstances, this vector can be grown and maintained in bacteria in LB + amp + str media. If you clone your gene of interest into the MCS and select for colonies that contain your recombinant plasmid, you can be confident you successfully transformed bacteria if: A. you see colonies growing on LB + amp + str plates. B. you see colonies growing on LB + amp plates but are str S. C. you see colonies growing on LB + str plates but are amp S. D. you see colonies growing on LB plates without antibiotics. E. none of the above
Ceyda G.
From the list below, select the sequence of steps for cloning a piece of foreign DNA into a plasmid vector, introducing the plasmid into bacteria, and verifying that the plasmid and the insert are present: (1) Transform competent cells (2) Select for the lack of antibiotic resistance gene #1 function (3) Select for the plasmid antibiotic resistance gene #2 function (4) Digest vector and foreign DNA with EcoR1, which inactivates antibiotic resistance gene #1 (5) Ligate the digested DNA together with the foreign DNA $a.$ 45132 $b.$ 45123 $c.$ 13425 $d. $32145 $e.$ 13254
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