Plasmid mapping. Plasmid mapping. QUESTION 1
A plasmid was cut with a range of restriction enzymes to produce different sized fragments. These fragments were run out on a gel and the fragment sizes produced are shown below.
Single restriction enzyme digests:
| Restriction enzyme |
Fragment sizes produced |
| EcoRI |
2670 |
| ScaI |
2670 |
| SapI |
410, 2260 |
| SspI |
740, 940, 990 |
Double digests Restriction enzymes
| Restriction enzymes |
Fragment sizes produced |
| EcoRI and ScaI |
2120, 550 |
| EcoRI and SapI |
410, 1130 |
| EcoRI and SspI |
420, 570, 740, 940 |
| ScaI SapI |
410, 580, 1680, |
| ScaI sspI |
130, 740, 810, 990 |
| SapI sspI |
180, 230, 560, 710, 990 |
Triple digest
| Restriction enzymes |
Fragment sizes produced |
| EcoRI and ScaI and SapI |
410, 550, 580, 1130 |
Use this information to re-construct a genetic map of the plasmid. Draw this plasmid map clearly for assessment. Be sure to include the exact base positions of all restriction enzymes and label your plasmid. Orientate the plasmid with the EcoRI restriction site at the top of the diagram (at base position 0).
QUESTION 2
Use your plasmid genetic map to answer the following questions
i) What would the shortest distance be between the EcoRI and ScaI restriction sites?
ii) What would the fragment sizes be if you digested the plasmid with EcoRI and SspI and ScaI?
iii) The Lac Z gene begins 150bp away from the EcoRI locus and 700bp from the ScaI locus. What would the distance be from the beginning of LacZ to the nearest SspI locus?
QUESTION 3 Research and explain the structure of the following plasmid features. Explain the reason for their inclusion in a plasmid used in molecular biology applications.
i) M13 locus
ii) T7 promotor
iii) LacZ iv) pUC origin
v) ampicillin resistance ORF
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