Restriction Enzymes and How to Use a Pipette

 Restriction Enzymes and How to Use a Pipette

1. Sticky Ends

Sticky ends are created when restriction enzymes cut DNA in a staggered manner, leaving single-stranded overhangs.

• Example Enzyme: EcoRI (recognizes GAATTC).
• Cutting Pattern:
  • DNA Sequence:
    5’ - GAATTC - 3’
    3’ - CTTAAG - 5’
  • Cut: After G on the top strand and A on the bottom strand, creating:
    • Sticky Ends:
      • 5’ - G AATTC - 3’
      • 3’ - CTTA A - 5’
• Uses: These overhangs can pair with complementary sticky ends, making them ideal for recombinant DNA technology.

Key Points in Diagram:

• Sticky ends are labeled as overhangs at “GAATTC.”
• They are used to insert complementary DNA sequences for genetic engineering or cloning.

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2. Blunt Ends

Blunt ends are produced when restriction enzymes cut DNA straight through both strands without overhangs.

• Example Enzyme: SmaI (recognizes CCGG).
• Cutting Pattern:
  • DNA Sequence:
    5’ - CCCGGG - 3’
    3’ - GGGCCC - 5’
  • Cut: Between the G and C on both strands:
    • Blunt Ends:
      • 5’ - CCC    GGG - 3’
      • 3’ - GGG    CCC - 5’
• Uses: Blunt ends are less specific for pairing but are more stable for certain molecular applications.

Key Points in Diagram:

• Blunt ends are shown as “cuts straight down.”
• They are described as “more permanent” since no overhangs are present for complementary pairing.

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Applications of Restriction Enzymes

1. Genetic Engineering:

• Enzymes like EcoRI and BamHI are used to cut and insert genes into host DNA.

3. How to Use a Pipette

Pipetting is a fundamental technique in molecular biology, used for accurately transferring small liquid volumes.

Types of Pipettes:

1. Micropipettes – Used for volumes between 0.1µL and 1000µL (1mL).

Steps to Use a Micropipette:

1. Select the Correct Pipette and Tip:

• P10 (0.5–10µL), P100 (10–100µL), P1000 (100–1000µL).
• Attach the correct disposable pipette tip securely.

2. Set the Volume:

• Adjust the dial on the micropipette to the desired volume.

3. Pipetting Technique:

1. Press the plunger to the first stop (before placing it in liquid).
2. Immerse the tip into the liquid (slightly below the surface, not too deep).
3. Slowly release the plunger to draw up liquid (avoid bubbles).
4. Transfer to the desired container, pressing the plunger to the second stop to dispense completely.
5. Eject the tip into a waste bin using the tip ejector button.