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11.5: Exercise 3 - Analyze the restriction digests on agarose gels

  • Page ID
    17563
    1. Plan your gel. Each group of students will prepare one agarose gel with 8 sample lanes.

    • Each student will run one lane with undigested plasmid and a second lane with plasmid that has been digested with RE. It is important to run a lane with undigested plasmid to determine if the REs have effectively cleaved the plasmids. Keep in mind that undigested plasmids will electrophorese with anomalous sizes because of their supercoiled structures.
    • One lane should be reserved for molecular weight standards.
    • Record in your notebook how you plan to load your gel.

    2. Prepare a 1% agarose gel in TAE buffer as described in Chapter 8.

    3. Prepare the samples for electrophoresis. Use the entire restriction digest on the gel. For undigested plasmid samples, set up tubes containing 7 μL plasmid and 3 μL deionized water.

    IMPORTANT: Return any unused plasmid to the freezer. You will use the plasmids for yeast transformation in a later lab.

    Combine:

    10 μL restriction digest or undigested plasmid

    5 μL deionized water

    3 μL 6X sample buffer

    4. Load and run the agarose gel as described in Chapter 8.

    5. Estimate the DNA concentrations in your plasmid preparations. After the gel has been run and stained, attempt to estimate the DNA concentrations in your three plasmid preparations, using the size standards as a reference. Each of the markers, with the exception of the 3 kb marker, contains ~40 ng. The 3 kb band contains 125 ng. Use these values to visually estimate the amount of DNA in your lane. Correct for the volume of sample in the lane to calculate the concentration of DNA in each plasmid preparation. (Although imprecise, this value will be useful for calculating your transformation efficiency in the next lab. )