Forensic Lab Procedures and Safety Practices

Review of Basic Safety Practices

Personal Protective Equipment (PPE) Usage

  • Minimizes contamination of forensic samples with biological agents.

  • Ensures the safety of personnel when handling blood, body fluids, or other potentially infectious materials.

Decontamination & Disinfection Protocols

  • Commonly used disinfectants: 10% bleach, alcohol-based solutions.

  • Prevents cross-contamination between forensic samples.

  • Reduces the risk of exposure to pathogens.

Proper Handling and Disposal of Biological Materials

  • Ensures safe handling, storage, and disposal of forensic samples.

  • Prevents accidental exposure to infectious agents.

  • Ensures that biological materials are disposed of in accordance with health regulations.

Aseptic Techniques

  • Samples are handled in a sterile manner to prevent contamination.

  • Protects DNA and other forensic materials, particularly when working with trace evidence.

Proper Handwashing

  • Duration: 40-60 seconds.

  • Required for soiled hands.

  • Important for dealing with C. difficile and B. anthracis.

  • Apply friction for 15 seconds when rubbing hands.

  • Rinse hands in a downward position.

Donning of PPE

  1. Perform Hand Hygiene

    • Hand wash for 40-60 seconds.

    • Hand rub for 20-30 seconds.

  2. Wear Laboratory Gown

    • Close the laboratory gown properly.

  3. Put on the face mask

    • Must cover nose and mouth.

    • Adjust the metal band to fit snugly over the nose.

  4. Put on Head Cap/Cover

    • Ensure no visible hair is seen outside the cover.

  5. Put on gloves

    • Pulled over the gown cuff

Doffing of PPE

  1. Remove Gloves

    • Follow procedure on proper removal of gloves.

  2. Remove Laboratory Gown

    • Follow proper procedure on removal of gown.

  3. Remove head cap/cover

    • Fish the head cover with the index fingers, make sure not to touch the outside part

  4. Remove Mask

    • Touching the string only

  5. Perform Hand Hygiene

Quality Assurance

Pre-Laboratory

Laboratory Phases

  1. PRE-ANALYTICAL

    • SPECIMEN HANDLING

      • Collection of Specimen

      • Transport of Specimen

    • DNA extraction

    • Quality Assessment

      • Purity and quantity of the extracted Nucleic Acid

    • Nucleic Acid Amplification

      • Polymerase Chain reaction (PCR) method

  2. ANALYTICAL

    • Detection and Analysis

      • Fluorescence, Gel electrophoresis, sequencing

    • STANDARDS

      • Cross-contamination of specimens must be avoided; this occurs mostly from pipetting carryover.

      • Aliquots removed from a specimen are never returned to the original tube or vessels.

  3. POST-ANALYTICAL

    • Results from electropherograms, gel images, and autoradiograms should be of sufficiently high quality.

      • Clear bands

      • Peaks without high background

      • No cross hybridization

      • No distortions

Pipetting and Measurement

Types of Measurements Used in the Laboratory

  • Volume

    • Units: mL, uL, L

    • Used for measuring the amount of liquid.

    • Applications: Preparing solutions, titrations, and transferring liquids.

  • Mass

    • Units: g, mg, kg

    • Used for measuring the amount of solid substance.

    • Applications: Preparing solutions.

  • Concentration

    • Units: % concentration

    • Applications: Titration, spectrophotometry

    • Definition: Amount of solute/amount of solution

  • Temperature

    • Units: °C

    • Importance: Precise temperature control is vital for enzymatic reactions.

  • pH

    • Scale: pH scale: 0-14

    • Indicates the acidity and alkalinity of the solution.

    • Applications: Chemical/biological experiments.

  • Time

    • Units: sec, min, hr

    • Tools: stopwatches, timers, clocks

    • Applications: Experiments involving rate of reaction, incubation time, or process time.

Types of Pipettes Used in the Laboratory

Proper Pipetting

  • Ensures the accuracy and precision of tests/experiments.

  • Consistency of results.

  • Eliminates systemic errors (air bubbles, meniscus reading, inconsistent plunger pressure).

  • Leads to better test/experimental outcomes.

Common Errors in Pipetting

  • Air bubbles in the tip.

  • Using an incorrect pipette for the volume required.

  • Pipetting too quickly.

  • Inconsistent plunger pressure.

  • Not using proper techniques for viscous liquids.

Activity No. 2: Preparing Solutions

  1. Prepare 4 tubes labeled with 2%, 3%, 4%, and 5% respectively.

    • 2%

    • 3%

    • 4%

    • 5%

  2. The desired volume of all the tubes is 10ml.

    • 2%

    • 3%

    • 4%

    • 5%

  3. Using a mechanical pipette, dispense 0.1ml of colored solution in all 4 tubes.

    • 2%

    • 3%

    • 4%

    • 5%

  4. Pipette the correct volume of distilled water to complete the concentration (%) required on each labeled tube.

    • 2%

    • 3%

    • 4%

    • 5%

  5. Complete the data needed in the table:

    • Desired Concentration | Solute Volume

    • 2% | 0.1ml

    • 3% | 0.2ml

    • 4% | 0.3ml

    • 5% | 0.4ml

Formula for Calculations

  • % \text{ Conc.} = \frac{\text{Solute}}{\text{TV}} \times 100

    • Where: TV = solute + solvent

PCR Amplification Procedure

  1. Add 50 ul of NSS on the extracted DNA.

  2. Mix by gently tapping microtube.

  3. Label PCR tube according to:

    • P1- group 1

    • P2- group 2

    • P3- group 3

    • P4- group 4

    • P5-group 5

  4. Label PCR tubes as follows:

    • (-) Negative Control

    • (+) Positive Control

  5. PCR reactions should be prepared as follows:

    • Add 20 ul Primer Mix (yellow)

    • Add 5 ul DNA sample (-/+, P1,P2,P3,P4,P5)

    • Add 5ul PCR Edvobead plus

  6. Mix each PCR sample. Make sure the PCR evobeads plus are completely dissolved.

    • NOTE: double check that both the primer mix and cDNA have been added by looking at the color of the mixture in the PCR tube. COLOR: orange with the primers and cDNA mixed together.

  7. Centrifuge the sample for a few 20 seconds to collect the sample at the bottom of the tubes.

  8. Amplify the DNA using the PCR machine with the following conditions:

    • Initial denaturation: 94°C for 3 minutes

    • Cycle (30 times):

      • 94°C for 30 secs

      • 55°C for 30 secs

      • 72°C for 65 secs

    • Final extension at 72°C for 4 minutes

Paper Simulation of Reverse Transcription

  1. Select a primer strip, prepare by folding it once along the dotted black line. Primer sequences are on the outside

  2. Find the complementary RNA sequence and match your primer sequence

  3. Find the first blank box immediately to the left of the primer sequence.

  4. Find your reverse transcriptase. Use the RNA sequence on the separate sheet and fill in the box with the complementary base pairing rules Write in a DNA nucleotide. Continue pairing and filling the boxes

  5. Unfold your prier strip. Continue writing the complementary base pairs in the empty boxes to the right of the forward primer.

Agarose Gel Electrophoresis

Procedure

  1. Prepare 1% agarose gel by dissolving 0.35g agarose in 30ml 1x TrisAcetateEDTA (TAE)

    • 29.5 ml of distilled water

    • 0.5 ml of concentrated buffer

  2. Mix the solution

  3. Microwave the solution on high for 1 minute. Carefully remove the flask from the microwave and mix by swirling. Continue heating in 15 seconds until agarose is completely dissolved

  4. Cool the agarose to 60°C by carefully swirling the flask to promote even dissipation of heat

  5. Add 35 ul SYBR Safe DNA stain into the solution. Swirl gently

  6. Pour mixture into the gel-casting tray and allow to harden. (20 minutes)

  7. Removed the combed.

  8. Place gel into the electrophoresis chamber. Cover the Gel with electrophoresis buffer

    • 147 ml distilled water

    • 3 ml Conc. buffer

    • Note: Gel should be completely submerged.

  9. Load the 25ul samples into the wells in a consecutive order

    • DNA ladder

    • (-)

    • (+)

    • P1

    • P2

    • P3

    • P4

    • P5

  10. Place safety cover. Check that gel is properly oriented.

  11. Connect the leads to power source. Perform gel casting tray from the electrophoresis chamber. (100volts for 30 minutes)

  12. Remove the casting tray with the gel and slowly slide the gel into the viewing surface of the transilluminator.

  13. Turn the unit On. DNA should appear as bright green bands on a dark background photograph units.

Electrophoresis

Definition

  • Movement of molecules like DNA, RNA, or protein mobilized by an electric field through a substance like agarose gel.

GEL Electrophoresis

  • Visualizes DNA and determines the sizes of the DNA.

  • Set up with 2 electrodes: negative and positive.

  • A method whereby charged molecules in a solution, chiefly proteins and nucleic acids, migrate in response to an electric field.

Laboratory Activity 4 Procedure

  1. Measure 350 ml of distilled water using the cylinder. Place the measured H2O in the beaker. Then add gelatin powder. Stir the solution until the powder dissolves.

  2. To make sure the homogenous consistency of your Gel add medium heat, DO NOT BOIL the solution.

  3. Pour your Gel solution into the molder and Leave the GEL at room temperature for at least 5 minutes.

  4. Remove the comb vertically, avoid moving or pulling the comb sideways, it may cause irregular well formation of the Gel.

  5. Transfer the molder containing the gelatin into the center of the GEL Electrophoresis equipment.

  6. Pour Distilled water at the side of the Gel Electrophoresis equipment make sure that the gelatin is submerged with water.

  7. Load samples into the wells using micropipette. For small well, pipet 5ul of colored solution. Use 10ul for bigger well. Colored solution must settle within the well.