Question

colony which one express gene

CORI -Hindi mal kpn ! Aggi -Hind III Gene CDNA EcoRI 5. G' AAT TC 3 CTTAAG. 5 Бра 5GGT ACC 3 3 CCATGG 5 pMAE2 Avr 11 M5 CCTAGGS 3 GGATCC.5 Arell ACCGGT 3 3.. TGGCCA...5 Amp Hind 1 5: AAGCTT 3 S 3. TTCGAA53 Xma C CGGG.3 GGGCGC 5 You try the following strategies in order to see if it work in order to transfer Gene X to the PMAE2 vector Strategy Restriction enzyme(s) used to cut... Gene X cDNA Plasmid vector EcoRI ECORI Hind III Hind III EcoRI & Nde I EcoRI & Nde1 Xmal Age 1 EcoRI & Age I EcoRI & Xma 1 EcoRI & Hind III EcoRI & Age 1 Xmal & Hind III Age 1 & Hind III KpnI & Hind III Hind III You next transform E. coli bacterial cells with a mixture of plasmids and the Gene X cDNA that you have digested with the restriction enzymes (strategy A-F). You plate the transformed cells on medium containing ampicillin and allow the bacterial cells to grow and form colonies. From the plate you select two transformed colonies (Colony A & Colony B) that have the Gene X cDNA insert. You realize that the gene could have been inserted into the plasmid in two different orientations. So you isolate the recombinant plasmids from two colonies, digest them with restriction enzyme M, resolve the digested fragments by DNA gel electrophoresis, and obtain an approximate profile of the DNA as shown on the next page.

Only correct explanation not just answears pls.
how to determine correct orientation by electrophoresis

Answer 1

The vector and insert together, regardless of the orientation of insert have only two restriction sites for Enzyme M, one is within the insert and the other is 3' to the MCS.

Now, the insert has one site, which is 0.5 kb from the 5' end of the insert and 1.5 kb from the 3' end. When the insert is in the correct orientation, that is the 5' end is towards the left and the 3' end is to the right, the digestion of the vector and insert plasmid would result in a fragment that is 1.5 kb plus some length, as the M site on the vector is 3' to the insert position. Therefore, Colony A contains the insert in the correct orientation.