Practical Final Revision – Pipetting, DNA Techniques, Electrophoresis & Karyotyping

Question-and-answer flashcards covering micropipetting technique, DNA extraction and PCR, electrophoresis principles, and human karyotyping with chromosomal abnormalities. Designed for practical final revision at New Mansoura University.

What are the two main types of automatic micropipettes?

Fixed-volume pipettes and variable (adjustable)-volume pipettes.

Why are automatic pipettes used in the laboratory?

To aspirate and dispense small liquid volumes accurately and reproducibly.

Which tip color is used for 0.5–20 µL volumes?

White tips.

Which tip color is appropriate for 20–200 µL volumes?

Yellow tips.

Which tip color allows pipetting of 100–1000 µL?

Blue tips.

What is the first step in proper micropipette use?

Select the required volume (remember 1 mL = 1000 µL).

At which stop is the plunger pressed before aspirating liquid?

The first (soft) stop.

Why should the pipette be held vertically during aspiration?

To avoid volume error and air entry.

At what angle should the tip be held during liquid delivery?

Approximately 20–45° to the container wall.

Which plunger stop is used to expel the full measured volume?

The second (hard) stop.

How do you avoid a loose tip error?

Load the tip with light but firm pressure until it seals correctly.

What problem results from quick plunger release, and how is it avoided?

It causes inaccurate volume and bubbles; release the plunger slowly.

Why must the plunger NOT be depressed to the second stop before immersion?

Doing so leads to over-aspiration; depress only to the first stop before dipping into the sample.

What is the correct immersion depth of the tip in the sample?

Just adequately below the liquid surface (about 1 cm) without touching the container bottom.

Define DNA extraction.

A routine procedure to isolate and purify DNA from biological samples for molecular or forensic analysis.

What is the principle behind spin-column DNA purification?

DNA binds to a silica membrane in the presence of chaotropic salts at specific pH, allowing selective retention and washing.

List six common components of a commercial DNA extraction kit.

Proteinase K, lysis solution, wash buffer I, wash buffer II, elution buffer, spin columns with collection tubes.

What are the four basic steps of spin-column DNA extraction?

Lysis, binding, two wash steps, and elution.

What is the role of Proteinase K in DNA extraction?

It digests protein components of cellular and nuclear membranes, aiding lysis.

Why is ethanol added during DNA extraction?

It promotes DNA precipitation and binding to the silica membrane.

How is DNA purity commonly assessed?

By agarose gel electrophoresis to check integrity and UV spectrophotometry (e.g., NanoDrop) to measure A260/A280 ratios.

Name the two major DNA amplification techniques highlighted in the notes.

Polymerase Chain Reaction (PCR) and cloning of DNA.

What are the five essential PCR reaction components?

Target DNA, a pair of primers, dNTPs, thermostable DNA polymerase (e.g., Taq), and buffer solution.

At what temperature does PCR denaturation occur, and what happens?

About 95 °C; double-stranded DNA separates into single strands.

During PCR, what temperature is typical for primer annealing?

Around 55 °C (exact temperature depends on primer Tm).

Which temperature is used for extension in PCR, and which enzyme acts?

Approximately 72 °C; Taq DNA polymerase synthesizes new DNA.

Roughly how many DNA copies are produced after 30 PCR cycles?

About one billion copies.

How is a PCR product usually checked after amplification?

By agarose gel electrophoresis to confirm fragment size.

Give two medical diagnostic applications of PCR.

Detection of infectious agents (e.g., HBV, HCV, COVID-19) and prenatal genetic diagnosis using amniotic or chorionic samples.

How is PCR used in forensic science?

For genetic fingerprinting at crime scenes and paternity testing from minute DNA samples.

State two additional applications of PCR beyond diagnostics.

Tissue typing before organ transplantation and studying evolution from archaeological DNA samples.

Define electrophoresis.

The movement of charged particles under an electric field; cations migrate to the cathode, anions to the anode.

Why does DNA migrate toward the anode during electrophoresis?

Because DNA’s phosphate backbone carries a negative charge.

Name two matrix types used in gel electrophoresis.

Agarose and polyacrylamide gels.

Which gel type is horizontal and best for separating large DNA fragments?

Agarose gel electrophoresis.

Which gel type provides high-resolution separation of short DNA fragments differed by only one nucleotide?

Polyacrylamide gel electrophoresis (PAGE).

List two properties of agarose that make it suitable for electrophoresis.

It is liquid when hot and solid on cooling, and forms a porous matrix allowing DNA migration.

What are the two purposes of adding bromophenol blue loading dye to a DNA sample?

It colors and increases density of the sample for easier loading, and allows tracking of migration during the run.

Which cable color connects to the negative pole on an electrophoresis apparatus?

Black cable to the cathode (negative pole).

What indicates that current is flowing properly in a gel electrophoresis tank?

The presence of bubbles at the electrodes.

What is a DNA ladder used for?

As a size standard to estimate the length of unknown DNA fragments by comparison.

How are DNA bands visualized after electrophoresis?

By staining with ethidium bromide and viewing under UV light (UV transilluminator).

Define a karyotype.

The complete set and appearance of chromosomes in an individual’s cell, typically shown during metaphase.

Why are cells arrested in metaphase for karyotyping?

Chromosomes are maximally condensed and aligned, making them easier to visualize and count.

Give three common indications for performing karyotyping.

Detect chromosomal abnormalities, determine gender, and investigate causes of birth defects, infertility, or genetic problems.

Name three sample sources that can be used for karyotyping.

Peripheral blood, amniocentesis/chorionic villus samples, or bone marrow/tissue biopsies.

Which chemical is used to arrest mitosis at metaphase during karyotype preparation?

Colchicine.

How is a normal male karyotype written?

46, XY.

What chromosome abnormality causes Down syndrome, and how is it written?

Trisomy 21; written as 47, XY + 21 for males or 47, XX + 21 for females.

State the karyotype of Turner syndrome.

45, X0 (female with a single X chromosome).

What additional chromosome is present in Klinefelter syndrome?

An extra X chromosome in males, giving karyotype 47, XXY.

Distinguish between numerical and structural chromosomal abnormalities.

Numerical involve extra or missing chromosomes; structural involve altered chromosome shape or arrangement (duplication, deletion, inversion, translocation).

List the four major types of structural chromosomal abnormalities.

Duplication, deletion, inversion, and translocation.