You have purified three different pSPORT1 plasmids pS1 pS2 a

You have purified three different pSPORT1 plasmids (pS1, pS2 and pS3) each with a different gene (A, B or C) cloned into it. You need to determine which plasmid has which gene. You know that the gene has been cloned into the plasmid so that EcoRI and Hindlll cut sites are near the ends of the gene. You instructor gives you EcoRI and Hindi III and asks you to determine the size of your genes. You digest all three plasmids with three different restriction enzyme solutions: E) Eco RI only, H) Hind III only and EH) Eco RI and Hind III together. After running your digest on an agarose gel you get the following result: Distances (mm) that the bands moved from the top for each lane in the gel above. You measure the distances of the bands in each lane (see table above). Using these distances you should: a) Make a standard curve from the market distances and calculate the size for the bands of unknown size in lanes 2-10 b) Determine the presence of internal EcoRI or Hind III cut sites in each. c) Determine the gene size for each plasmid d) Using the table below and your calculations above, identify the gene in each of the plasmids. Gene listing with size and the fragments that would be generated with the difference digests.

Solution

In PS1 the insert doesnot have EcoRI and Hind sites as digestion with a single enzyme just linearised the plasmid and produced the 5000bp band which is the size of the plasmid. Double digestion with EcoRI and Hind released the insert of size 1500 bp and produced the other fragment of size 400bp. Hence the gene insert size in PS1 is 1500bp which does not carry any restriction sites for enzymes.

In PSII and PSIII digestion need to be repeated as the bands are not as expected.