Dundalk Institute of Technology 7

Restriction Enzymes in DNA Analysis

• Overview of Restriction Enzymes

  • Used for cutting DNA into fragments for analysis.

  • Common examples:

    • EcorR1

    • HindIII

• Types of Restriction Enzymes

  • Each enzyme recognizes specific sequences in DNA and cuts at those points.

  • Example:

    • EcorR1: Cuts at a specific site, facilitating further analysis.

    • HindIII: Has a different recognition site from EcorR1.

Preparing DNA Samples

• Concentration Calculation

  • Objective: Achieve a concentration of 1 microgram per milliliter in final samples.

  • Steps for preparation:

    • Determine the current DNA concentration.

    • Divide this concentration into 1000 nanograms to calculate the required volume to achieve the desired concentration.

• Example Calculations

  • For instance, if the sample concentration is 1900nanograms:

    • To find volume needed for 1000 nanograms, divide by the current concentration.

  • Check various initial DNA concentrations (like 8060 or 800) to adjust for accurate preparation.

Practical Tips for DNA Preparation

• Using Nanodrop

  • Tool to measure DNA concentration accurately.

  • Recommended to take 4 microliters off for analysis to prevent over-extraction.

• Preparation Strategy

  • Have ample DNA samples available when performing digestion reactions to ensure enough to work with.