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4: Micro-report 3: Restriction mapping

  • Page ID
    17597
  • Figure: The figure at the heart of this micro-report is an agarose gel containing the restriction digests of your team’s plasmids. Refer to the guidelines for Micro-report 2 to see how to set up figures with agarose gels. The reader should be able to determine from the gel and its legend which plasmid is shown in each lane and which restriction endonuclease (RE) was used in each digest.

    Materials and Methods: To reproduce your experiments, a reader will need to know which plasmids and REs that you used, how you purified the plasmids and some information about the agarose gels used for the restriction maps. When possible, refer to published procedures, noting any modifications to a published procedure. You may want to use subheadings for the M&M section.

    Plasmids: The MET genes and homologs have been cloned into two different yeast overexpression plasmids. Use the correct nomenclature when referring to plasmids of known genotypes. Plasmid names begin with a lower case “p” and are written in normal font. For much of this report, you will need to refer to the plasmids by their number alone, since you are still trying to identify them.

    Plasmid purification: There are many different methods for isolating plasmids, so inform the reader that you used the ZyppyTM Plasmid Mimiprep Kit (Zymo Research) with the modifications described in the lab manual. Include information on how you estimated plasmid DNA concentrations as well as the DNA concentration of each purified plasmid.

    Restriction digests: To reproduce your experiments, the reader will want to know how much plasmid DNA you used in the digestions and which REs you used. The ratio of RE units to DNA is important for successful digests. Your reader may be using a different commercial source of REs, so also provide the number of enzyme units (U) in the reaction. Do NOT list the μL of each component used in the reactions. Be sure to include information about the temperature and duration of the incubation.

    Agarose gel electrophoresis: See the guidelines for micro-report 2.

    Results and Discussion: Bring the reader through the logic for your experimental design. Why did you choose this particular RE? Refer to the bands on the gels as restriction fragments. Restriction fragments are the products of RE digestions.

    Size is very important in interpreting the results of a RE digest. A single summary data
    table with the predicted and observed sizes of the restriction fragments should be included. Estimate the sizes of the restriction fragments by comparing their migration to the markers. Predicted sizes should very accurate, since they are based on the actual DNA sequences, which has nucleotide resolution. Your estimated sizes are much less accurate. You may be able to determine if one restriction fragment is smaller or larger than another, but you will only be able to place the fragments within a certain size range. NOTE: circular plasmids run anomalously on agarose gels. Supercoiled plasmids migrate more rapidly on gels than a linear DNA molecule of the same size. Conversely, nicked (a single strand of the helix has been cleaved, producing a relaxed circle) plasmids migrate more slowly than linear DNA of the same size. Incomplete digestion can also complicate results. Keep in mind that the number of bands on the gel is not as important as the differences in the banding patterns!

    Did your experimental results confirm your predictions? If your results did not confirm your predictions, you need to consider if the predictions were wrong OR if the reactions did not work. The undigested plasmid provides a control for interpreting the latter issue.