Plasmid Mapping 1. Overview of Plasmid Mapping Plasmid mapping is a technique used to determine the structure and organization of DNA within a plasmid, including the location and orientation of inserted DNA fragments. It involves: • Restriction enzyme digestion. • Gel electrophoresis to visualize DNA fragments. • Analysis of band patterns to infer DNA fragment sizes and orientations. 2. Insert Orientation When inserting DNA fragments into plasmids, the orientation can vary based on the restriction enzymes and ligation process. Inserts can ligate in two possible orientations: • Correct orientation (5′ → 3′ ) allows proper gene expression. • Incorrect orientation (3′ → 5′ ) may inhibit expression. Key Goal: Identify which plasmids contain the insert in the correct orientation. 3. Experimental Setup Plasmid Information: • Plasmid: ∼ 5.7 kb • Insert: ∼ 4.5 kb + 1.1 kb or ∼ 3.7 kb + 2.0 kb depending on orientation. Restriction Digests: • Enzymes: HindIII, BamHI, HindIII + BamHI. • Digests are run on a 0.75% agarose gel at 100 V for 30–45 minutes. • A 1 kb DNA ladder is used for size reference. • DNA is visualized with Red Safe staining under UV light. 1 4. Gel Electrophoresis Basics Principle: • DNA is negatively charged and migrates towards the positive electrode when subjected to an electric current. • Smaller DNA fragments migrate faster, while larger fragments travel more slowly. Agarose Gel Preparation: • Agarose forms a porous matrix; higher percentages provide better resolution for smaller fragments. • Red Safe dye intercalates between DNA bases and fluoresces under UV light, enabling visualization. Loading Dye: • Contains glycerol to ensure samples sink into wells. • Includes tracking dyes (e.g., bromophenol blue) to monitor gel progress. • EDTA chelates metal ions, halting enzymatic activity. 5. Types of DNA Bands on the Gel • Uncut Plasmid: Appears in multiple forms: – Supercoiled. – Nicked. – Linear. • Single Digest: Plasmid is cut once, producing a single linear band. • Double Digest: Plasmid is cut twice, resulting in two bands. These bands are critical for determining insert orientation. 6. Practice Problem: Determining Insert Orientation Scenario: You perform a double digest of your plasmid with HindIII and BamHI. The gel shows the following band sizes: • Band 1: 3.7 kb. 2 • Band 2: 2.0 kb. Question: What is the orientation of the insert? Solution: • Based on the data, the 5′ → 3′ orientation produces bands of 3.7 kb and 2.0 kb. • The insert is in the correct orientation for expression. 7. Importance of Controls Undigested Plasmid: • Serves as a control to ensure plasmid integrity. • Identifies supercoiled, nicked, or concatameric forms. Single Digest: • Ensures the enzyme activity is optimal. • Troubleshoots incomplete digestion (e.g., bands matching undigested plasmid). 8. Reporting Results • Label each lane and identify band sizes using the DNA ladder. • Use band sizes to determine insert orientation. • Discuss how insert orientation affects gene expression levels in your lab report. 3