Assignment
Cloning By Computer

Go through the "Recombining DNA On The Computer" exercise.

Submit a report on the exercise which includes the following information:

1. Modified A chain DNA sequence (see Part III, Step 4) (20 pts.).
2. Sequence of the recombinant pUC19 plasmid (i.e. plasmid with the modified A chain DNA sequence (see Part IV, Step 3) (20 pts.).
3. Diagram of the non-recombinant pUC19 plasmid showing the location (base pair number) of the EcoRI and BamHI cleavage sites, and the total length of the plasmid (see Part IV, Step 2) (15 pts.).
4. Diagram of the recombinant pUC19 plasmid showing the location (base pair number) of the EcoRI and BamHI cleavage sites, and the total length of the plasmid (see Part IV, Step 4) (15 pts.).
5. Diagram of an agarose gel showing the expected migration positions of DNA fragments obtained following cleavage of pUC19 (lane 1) or recombinant pUC19 (lane 2) with EcoRI and BamHI. Indicate the expected sizes of the DNA bands in base pairs, and include which electrodes go at which ends of the gel (15 pts.).
6. Amino acid sequence of the insulin A chain as expressed in bacteria using your recombinant plasmid (see Part V) (15 pts.).

Total value of exercise: 100 pts.

You may submit the completed assignment by the usual means.

 NOTES:

• The diagram of the pUC19 plasmids, both unmodified and recombinant (items #3 and #4) can be represented by a hand-drawn or computer-drawn drawing.
• Your results from the amino acid sequence (item #6) will NOT agree with the sequence given on page 125 of your textbook The sequence in your textbook is that from bovine insulin, whereas you are using human insulin in your lab. Use your data from this lab and do not double-check it against the textbook.
• Remember that protein sequences start with a certain codon and end with one of three possible codons. Codons before the start codon or after the stop codon are not translated.