DNA and RNA Measurement Techniques

Labs - Molecular Biology

• Measuring weights uses high-accuracy balances. Units like milligrams (mg) are common.
• Preparing stock solutions: combine a substance with distilled water, known concentration and volume, then calculate the mass using the formula: C = n/V (where C is concentration, n is moles and V is volume)
• Balance checks: Level indicator bubble should be centered for accurate measurements.
• Before using a balance, always check its capacity and position the container in the middle.
• Temperature control: Samples should be at room temperature. Some substances evaporate quickly, so they must be weighed quickly.
• Balance usage tips: Always use the same balance for all readings, and check balance quality with a known mass.
• Solution preparation: Solutions should be labeled with substance name, concentration, and date.
• Volumetric flask: used for accurate volume measurements. The meniscus (curved surface of the liquid) must be at the calibration mark. Rinse tools to avoid losing solution and causing errors.

Measuring Volumes

• Large volumes: Use graduated cylinders for volumes up to 2 liters, or volumetric flasks for high accuracy.
• Microliter range: Use micropipettes; there are different micropipette types (P10, P100, P1000) for different ranges of volume measurements, with special attention on calibrating the accurate measurements scale, to avoid errors.
• Avoid using beakers or flasks for precise volume measurements.

Micropipette details

• Micropipette components: plunger button, friction ring, digital volume indicator, shaft, tip ejector, and tip.
• Micropipette types and ranges: P10 (1-10µL), P100 (20-100µL), P1000 (100-1000µL). Choose the right pipette based on the volume needed.
• Micropipette usage: Follow the steps: check the pipette's range; attach the tip; press the soft plunger; dip the tip into the liquid and slowly release; move the substance to the desired place and slowly press; and press hard to avoid losing anything; adjust volume to max value; avoid damaging the spring.
• Use a spinner to lay substances down at the bottom of a tube. Ensure the balancing of the substances, using accurate balancing.

Lab 4: DNA Extraction

• DNA protection mechanisms: Prokaryotes lack a nucleus and nuclear membrane, while eukaryotes are surrounded by a membrane-bound nucleus; plants have additional cell wall protection. DNAse enzymes in the cytoplasm must be denatured before extracting DNA.
• Cell lysis: Bead beating and vortexing with phenol break the cell wall; detergent (SDS) removes the lipid membrane.
• Precipitation of protein: Adding salt (like NaCl or NaCH3COO) removes remaining proteins, and vortexing with phenol-chloroform and centrifuging separates nucleic acids from other substances.
• DNA precipitation: Use cold ethanol or isopropanol; wash with cold alcohol to prevent DNA denaturation; DNA re-suspension in TE or Tris.
• Extraction using the Phenol-Chloroform method: add sample to an eppendorf tube, add NaPO₄ & TNS, use freeze-thaw method to break cell wall, add proteinase K and incubate, centrifuge and separate supernatant. Take amount of supernatant, add phenol/chloroform/isoamyl alcohol, vortex, centrifuge, centrifuge the tube, add 70% ethanol, vortex, and centrifuge again, and get the pellet to dry, adding TE buffer and store in -20°C.

Lab 5: Plasmid Extraction

• Plasmids are extrachromosomal DNA molecules that confer survival advantages, like antibiotic resistance, to bacteria.
• Plasmid isolation: Discard supernatant, resuspend cells with the resuspension solution in a vortex, transfer to a centrifuge, add lysis solution, invert gently, add neutralization, invert again to disrupt the bacterial chromosome.

Lab 6: DNA Quantification

• DNA sample preparation: Dilute DNA by mixing 10 mL of DNA with 990 µL of distilled water for a 1:100 dilution.
• Spectrophotometer setup/use: Warm the spectrophotometer for 15 minutes with no cuvette inside; measure absorbance at 260 nm (A260), 280 nm (A280). A260/A280 ratio must be greater than 1.8-2.0, this shows purity.

Lab 7: Restriction Digestion

• Restriction enzymes: Enzymes that cut DNA at specific sequences. Palindromes are short sequences read the same backward.
• Tips for restriction digestion: Use buffers from different companies if compositions are the same; enzyme volume should be 1/10 of the total reaction volume; incubation time longer than 1 hour but not overnight. Use uncut DNA along with digested DNA, and a DNA marker for proper gel analysis.

Lab 8: Agarose Gel Electrophoresis

• Method to separate DNA/RNA fragments by size. Negative DNA moves toward the positive electrode.
• Agarose gel advantages: Easy pouring, no effect on DNA, and easy sample recovery.
• Agarose gel disadvantages: Melts at high temperatures, buffer changes are needed, and some materials' behaviors are unpredictable.
• Applications: Estimating DNA size, PCR product analysis, and separating restricted genomic DNA.

Lab 9: Factors affecting DNA migration

• DNA length: Shorter fragments migrate faster than longer ones.
• Agarose concentration: Higher concentration separates smaller molecules better, while lower concentration separates larger molecules better.
• Voltage: Higher voltage speeds up migration but reduces resolution (clarity of separation).

Lab 10: DNA Sequencing (Sanger Method)

• Method for determining the precise order of nucleotides in a DNA molecule.
• Techniques include PCR, electrophoresis, and analysis of resulting fragments.
• Manual sequencing involves separate tubes for each dideoxynucleotide (ddNTP) to halt chain elongation at specified points, showing different fragments.
• Automated sequencing uses a single tube with labeled ddNTPs, allowing detection and sequencing of many different nucleotide fragments in the same run.

Lab 11: DNA Sequencing (Automated Sanger)

• Using single tube with all ddNTPs, with fluorescent labels allowing detection.

This quiz focuses on critical aspects of measuring purity and concentration in DNA and RNA analysis. It covers key concepts related to absorbance, wavelength significance, and best practices for equipment placement. Test your understanding of the principles associated with optical density and measurement accuracy.