Molecular Biology Protocols

🧪DNA Purification Protocol

Molecular biology protocols begin with effective DNA purification. This essential technique ensures high-quality genetic material for downstream applications.

Materials Required:

• DNA extraction kit (Qiagen DNeasy or equivalent)
• Microcentrifuge tubes (1.5 mL)
• Pipettes (10-1000 μL range)
• Vortex mixer
• Microcentrifuge

Step-by-Step Protocol:

1. Collect 200 μL of sample in sterile microcentrifuge tube
2. Add 20 μL proteinase K and mix gently by vortexing
3. Add 200 μL Buffer AL and incubate at 56°C for 10 minutes
4. Add 200 μL ethanol (96-100%) and mix thoroughly
5. Transfer mixture to DNeasy Mini spin column
6. Centrifuge at 6,000 x g for 1 minute, discard flow-through
7. Add 500 μL Buffer AW1, centrifuge at 6,000 x g for 1 minute
8. Add 500 μL Buffer AW2, centrifuge at 20,000 x g for 3 minutes
9. Elute DNA with 50 μL Buffer AE, incubate for 1 minute
10. Final centrifugation at 6,000 x g for 1 minute

Expected Yield: 5-15 μg of high-purity DNA

Quality Control: A260/A280 ratio should be 1.8-2.0

Reference: Qiagen Protocol Database

🔬Plasmid DNA Transfection Protocol

Plasmid transfection represents a crucial molecular biology protocol for introducing genetic material into mammalian cells.

Materials Required:

• Plasmid DNA (1-5 μg, endotoxin-free)
• Transfection reagent (Lipofectamine 3000)
• Opti-MEM reduced serum medium
• Cell culture plates (6-well or 24-well)
• Complete growth medium

Transfection Protocol:

1. Seed cells at 70-90% confluence 24 hours before transfection
2. Dilute 2.5 μg plasmid DNA in 125 μL Opti-MEM
3. Add 3.75 μL P3000 reagent to DNA solution, mix gently
4. Dilute 3.75 μL Lipofectamine 3000 in 125 μL Opti-MEM
5. Incubate both solutions separately for 5 minutes
6. Combine DNA and lipofectamine solutions, mix gently
7. Incubate DNA-lipid complex for 15 minutes at room temperature
8. Add 250 μL complex dropwise to cells in fresh medium
9. Incubate cells at 37°C, 5% CO2 for 24-48 hours
10. Analyze transfection efficiency by fluorescence or Western blot

Expected Efficiency: 60-90% in most cell lines

Optimization Tip: Adjust DNA:lipid ratio for different cell types

Reference: Thermo Fisher Transfection Guide

📊Flow Cytometry Protocol

Flow cytometry enables precise cellular analysis in molecular biology protocols, providing quantitative data on cell populations.

Sample Preparation:

1. Harvest cells by trypsinization or gentle scraping
2. Wash cells twice with PBS containing 2% FBS
3. Resuspend cells at 1 x 10⁶ cells/mL in staining buffer
4. Aliquot 100 μL cell suspension per tube
5. Add primary antibodies at recommended dilutions
6. Incubate for 30 minutes at 4°C in darkness
7. Wash cells twice with staining buffer
8. Add secondary antibodies if using indirect staining
9. Incubate for 30 minutes at 4°C in darkness
10. Final wash and resuspend in 300 μL PBS for analysis

Acquisition Parameters:

• Forward Scatter (FSC): Linear scale
• Side Scatter (SSC): Linear or log scale
• Fluorescence channels: Log scale
• Acquisition rate: 500-1000 events/second
• Total events: 10,000-50,000 per sample

Reference: BD Biosciences Flow Cytometry Protocols

🧬RNA Genetic Code Table

The genetic code serves as the foundation for all molecular biology protocols involving RNA analysis and protein synthesis.

Key Features:

• 64 codons encode 20 amino acids plus stop signals
• Code is degenerate (multiple codons per amino acid)
• AUG serves as universal start codon
• Three stop codons: UAA, UAG, UGA

Reference: NCBI Genetic Code Database

🔍Intracellular Staining Protocol

Intracellular staining protocols enable visualization of internal cellular components, essential for advanced molecular biology protocols.

Fixation and Permeabilization:

1. Harvest cells and wash with PBS
2. Fix cells with 4% paraformaldehyde for 15 minutes at room temperature
3. Wash twice with PBS to remove excess fixative
4. Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes
5. Block non-specific binding with 5% normal serum for 30 minutes
6. Wash cells once with PBS

Antibody Staining:

1. Incubate with primary antibody (1:100-1:1000 dilution) overnight at 4°C
2. Wash three times with PBS-Tween (0.05%)
3. Apply fluorescent secondary antibody (1:200-1:500) for 1 hour
4. Wash three times with PBS-Tween
5. Counterstain nuclei with DAPI (1 μg/mL) for 5 minutes
6. Final wash and mount with anti-fade medium

Imaging Parameters:

• DAPI: Excitation 358 nm, Emission 461 nm
• FITC: Excitation 495 nm, Emission 519 nm
• Texas Red: Excitation 595 nm, Emission 615 nm
• Use appropriate filter sets for each fluorophore

Reference: Abcam Immunofluorescence Protocols

📋Patient Care Report Narrative Examples

Documentation protocols ensure proper record-keeping in clinical molecular biology protocols and research applications.

Sample Report Structure:

Documentation Requirements:

• Patient consent and identification verification
• Chain of custody documentation
• Quality control results and interpretations
• Technician signatures and timestamps
• Storage conditions and sample tracking

Reference: CAP Laboratory Standards