Molecular Biology-2014
Assignment #3
Exercise 3
Restriction mapping and agarose gel electrophoresis
1. Define the following terms: Isoschizomer, neoschizomer, and isocaudomer.
2. Amongst the enzymes listed on page 37 of the lab manual, which, if any, generate compatible ends to each other? (Ex. Sau3aI and BamHI)
3. Submit a figure representing your agarose gel. Make sure to include an appropriate legend. Indicate in the figure legend the name of the gene of which you are verifying the restriction map. (Bioinfo exercise) See the directives on this course’s web site under the heading Figures/Graphs.
Bioinformatics 2
Restriction mapping
4. Present theoretical maps of all unknown genes. Indicate below each map the name of the gene of which you are verifying the map and list the enzymes which do not cut.
5. Present theoretical restriction maps (2 maps representing both possible orientations) of the gene you are verifying inserted within the multiple cloning site of pUC19. See the example on this course’s web page under the heading Figures/Graphs. Your map must include the following informations:
a. Multiple cloning site of pUC19 including restriction sites for all enzymes used in this lab.
b. The insertion site.
c. The theoretical positions, as determined from the bioinfo exercise, within the gene of the restriction sites for all the enzymes used in this lab.
6. Submit a table which compares the experimental data obtained following the analysis of your gel to the theoretical data obtained following the bioinformatic analysis. Your table must present the following data: Enzyme used, Number of cuts, and Fragment sizes resulting from complete digests. (do not include fragment sizes resulting from partial digests)
7. Based on your analysis in the previous question, submit a table which proposes potential double digests to confirm the orientation of your gene within the vector, and the position of any restriction site within the insert. Include the predicted sizes from these digests for both orientations.
Manipulating sequences
8. Present the complement, inverse, and inverse complement of the following sequence:
5’- GAATGCGGCTTAGACTGGTACGATGGAAC-3’
9. Provide a printout of each of the following derivatives of the mer sequence.
· Reverse sequence of template
· Complement sequence of the template
· Reverse complement sequence of the template
· Reverse sequence of the complement sequence of the template
· Obtain the complement sequence of the reverse sequence of the template.
Primer alignment using Blast
10. What is the predicted size of the LacZ amplicon?
11. Indicate the identity and position of the mismatch within the primer PCRmutagenGAA
Primer mapping
12. Submit a figure showing the positions and directions of each of the following primers on the pUC19 sequence.
A. TGCGGTGTGAAATACCCT
B. GCCATTCAGGCTGCGCAA
C. GGGTTATTGTCTCATGAG
D. GAGACAATAACCCTGATA
Indicate in a legend to your figure all primer pairs, if any, would give an amplification product of at least 200bp.
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