Restriction Mapping of Plasmid DNA

Restriction Enzymes and Restriction Mapping

• Bacteria use restriction enzymes to recognize and cut specific DNA sequences, helping defend against invading viruses.

• EcoRI cuts DNA to produce sticky ends with a 4-base overhang.

• HaeIII cuts to produce blunt ends.

• Restriction maps are built by comparing fragment sizes produced by different restriction enzymes.

• pAMP plasmid: small circular DNA (approximately 3,000–8,000 bp) used as a genetic vector.

Lab Objective (Restriction Mapping of pAMP)

• Construct a restriction map of the pAMP plasmid using BamHI, EcoRI, and HindIII.

• Analyze fragment sizes from enzyme digests via gel electrophoresis to locate restriction sites.

Gel Electrophoresis Basics

• DNA is negatively charged and moves toward the anode under an applied electric field.

• Smaller DNA fragments migrate faster than larger ones; separation by size.

• Molecular Weight (MW) Ladder provides fragments of known sizes for comparison.

• GelStar™ dye stains DNA so bands are visible under UV light.

• Gel images help determine fragment sizes when compared to the ladder.

Apparatus and Reagents

• Gel: 0.8% agarose in 1X TAE.

• 10X TAE solution prepared earlier; dilute to 1X for gel use.

• CutSmart Reaction Buffer: 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 µg/mL BSA, pH 7.9 @ 25°C.

• Gel casting components: gel tray, comb, power supply, GelStar™ dye, Quick-Load 1 kb ladder.

Digestion of pAMP (Overview)

• Prepare 5 tubes:

  • Tube 1: pAMP control (no enzyme).

  • Tube 2: pAMP + one enzyme (A).

  • Tube 3: pAMP + two enzymes (B).

  • Tube 4: pAMP + two enzymes (C).

  • Tube 5: pAMP + two enzymes (D).

• Include pAMP DNA, CutSmart buffer, diH2O, and respective enzymes in each tube.

• Incubate at 37°C for at least 1 hour.

• Store samples at 4°C for short-term or at -20°C for long-term.

Agarose Gel Preparation and Running

• Prepare 0.8% agarose in 1X TAE; cast in gel tray with comb for wells; insert GelStar™-DNA mix; allow to solidify.

• Fill gel box with 1X TAE so the gel is covered.

• Prepare tracking dye (4 µL per digestion tube) containing Ficoll®, EDTA, Tris-HCl, SDS, and dyes.

• Load 1 kb ladder in the first well; load 10 µL of each digestion sample in separate wells.

• Run: start at 70 V for 20–30 min, then 90 V for ~45 min or until the dye migrates about ¾ of the way through the gel.

• Remove gel and visualize under UV light; print an image for analysis.

Standard Curve and Fragment Sizing

• Principle: fragment migration distance is inversely related to fragment size.

• Use MW ladder to plot a standard curve: distance traveled vs. log(bp).

• Relationship: plotting distance (y) against log(bp) (x) yields a linear equation: $ext{log}_{10}( ext{bp}) = m \, y + b$

• To find fragment sizes: measure the migration distance of sample bands (y), then compute $ext{bp} = 10^{(m y + b)}$.

• The experiment yields two-fragment patterns from chosen enzyme combinations; base the map on these fragment sizes.

Determining the Restriction Map

• Compare observed fragment sizes (from each enzyme digest) to the MW ladder-derived sizes.

• Identify which two-enzyme combination (among BamHI, HindIII, EcoRI) produced two fragments.

• Use fragment sizes to infer relative positions of restriction sites on pAMP.

• Cross-check with the shown restriction map to determine the exact enzyme pair used by the group.