233 Hindill (1) 234 Hindlll (1) 415 EcoRV (2) 527 Styl (2) 767 Pstl (2) 942 Pstl

Question

233 Hindill (1) 234 Hindlll (1) 415 EcoRV (2) 527 Styl (2) 767 Pstl (2) 942 Pstl (1) 1017 BamHI (1) pAMP pKAN 4539 bp 4194 bp 1150 Styl (2) 1690 Pstl (2) 2094 EcoRV (2) 2137 EcoRI (1) 2116 EcoRI (1) 2095 BamHl (1) 384 EcoRV (1) pGLO 5371 bp 1239 BamHI (3) 1465 Hindlll (2) 1510 Styl (1) 1864 BamHI (3) 2063 EcoRI (1) 2084 BamHl (3) 2102 Pstl (2) 2114 Hindlll (2) 3177 Pstl (2) Figure 1. Restriction maps for pAMP, pKAN, and pGLO, three of the plasmids found in the company stocks of bacteria. The name of the plasmid and the total number of base pairs are shown in the middle of the circle. Restriction sites are shown. The number before the restriction enzyme name is the position of the site and the number in parentheses after the restriction enzyme name is the number of times that particular enzyme cuts the plasmid

Explanation / Answer

If you oberve the given plasmid maps of pAMP, pKAN and pGLO, we can say that EcoRI is the only enzyme that can cut all three plasmid only once.

- SO for the restriction digest expriment of the unknown plasmid, the control that will be used is

the reagents that all three control sample will contain are

- When the above mentioned digestion is perfomed with the control samples and run on a agarose gel, we get the exact size of the respective plasmid. SO pGLO when digested should run at approzimately 5 kb. Similarly pKAN at 4kb and pAMP at 4.5 kb.

By comparing the size of the restriction digestion product of the sample with the control, we determine its size and thus the identity of the plasmid.

- As mentioned earlier, EcoRI is the only enzyme that can cut all three of the plasmids only once. Hence this enzyme will be used to digest the sample and determine its identity.

- When the sample is digested with EcoRI and run on a gel along with the controls

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