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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