LAB EQUIPMENTS, MICRO-PIPETTING AND THE USE OF pH METER

Reagent preparation room

also known as pre-PCR activities

Sample preparation room

PCR room

includes PCR execution

Buffer

where the medtech will decontaminate to ensure that there are no contaminants that might go inside the areas

Work Table

where we will mix the reagents; calculate solutions, etc.

Pass Box

where we will put the prepared solution

1 Billion

A typical PCR generates as many as ____ copy of target sequence

1 Million

Aerosols from pipettes will contain as many as _____ amplification

products

CONTAMINATION IN A PCR LAB

• Amplification product contamination

Cross contamination between specimens - To avoid this, use different pipette tips

• Laboratory surfaces (e.g. fomites)

• Ventilation ducts (e.g. exhaust products or system, etc.)

• Reagents/supplies

• Hair, skin, saliva, and clothes of lab personnel (have DNA and RNA)

HOW TO CONTROL CONTAMINATION:

- Correct Laboratory Design – must be one-way

- Laboratory Practices – follow the laboratory rules such as no eating, drinking, etc. in the laboratory

• Chemical/Enzymatic Control

Chemical Control

Lysol, sodium hypochlorite, alcohol

Enzymatic Control

prevent unnecessary nucleic acids (e.g.: DNAse = remove DNA; RNAse = remove RNA)

Incorrect results

Require extensive clean-up

Loss of credibility

Impact on economy and performance

WHAT HAPPENS IF THERE IS LACK CONTAMINATION CONTROL?

BIOSAFETY CABINET

Protection to the user, product, and environment

Decontamination

10% Bleach→70% Ethanol

BIOSAFETY CABINET CLASS I

Sash/window is opened up to protect the use from the sample or specimen we’re working on

BIOSAFETY CABINET CLASS I

Unfiltered air enters and filtered air comes out

BIOSAFETY CABINET CLASS I

Protect the environment

• Does not protect the sample because of the unfiltered air passing through the specimen or sample

BIOSAFETY CABINET CLASS I

Air goes inside cabinet without filtration, comes out of cabinet with filtration

• No product protection needed

Biosafety Cabinet Class I

- Opening suspicious mail

- Running Centrifuges

- Housing fermenters

- Aerating cultures

BIOSAFETY CABINET CLASS II

• Better than BSC Class I

• Provides personnel, product, and environmental protection

BIOSAFETY CABINET CLASS II

• Does not provide absolute containment, can only provide if these 3 aspects are maintained: - Open front with inward airflow - Downward HEPA Filtered Laminar Airflow - HEPA Filtered Exhaust Air

Positive Pressure

Pushes Air

Negative Air

Pulls Air

TYPE A1

- Plenum is under positive pressure

- Some air will be exhausted and some will be recirculated

- Without Canopy/exhaust connecting to the outside of the facility

TYPE A2

- Plenum (air-filled space where air is distributed) is under negative pressure

- Two types:

1. Without Canopy

2. With Canopy (can minimize release of chemical fumes in the room)

- 60-70% is recirculated; 30-40% is exhausted

Type B1

- 40% Air circulated

- 60% exhausted outside thru hard ducting (HEPA filters)

Type B2

- 100% air exhausted via hard ducting (HEPA filters)

Type C1

- Similar mechanism with Type B, but more available and flexible mechanism

BIOSAFETY CABINET CLASS III

• Safest; used for higher level of BSL organisms such as Bacillus anthracis

• Gas tight, under negative pressure

BIOSAFETY CABINET CLASS III

• No recirculation of air

• HEPA Filtered air supply

BIOSAFETY CABINET CLASS III

• Exhaust air: Double HEPA-filtered or HEPA-Filtered and Incinerated

BIOSAFETY CABINET CLASS III

• Operation inside thru Rubber Gloves [built-in gloves that is also called as glove boxes]

BIOSAFETY CABINET CLASS III

• Cabinet connected to a double-door autoclave and/or chemical dunk tanks to sterilize/disinfect all exiting materials

DRY BATH

• Not commonly used in molecular biology laboratory. Instead, we use ice basket

• For heating/warming/boiling of samples

• Physical Inactivation

Digital

Analog

Two types of Dry bath

ELECTROPHORESIS APPARATUS

• PCR → needs electrophoresis

• DNA, RNA, Protein separation

• • Used for: - Detection - Purification

Anion

negatively-charged particles

Cation

positively-charged particles

Anode

Positive Electrode

Cathode

Negative Electrode

MICROCENTRIFUGE/MICROFUGE

Principle: Sedimentation by Centrifugal force

• For samples less than 2 mL

MICROCENTRIFUGE/MICROFUGE

Used to mix or separate particles in suspension, used for PCR techniques

• Speed: Up to 17000 x g

• Max samples: 24 per run

MICROPIPETTES

For volume measurements <1000 uL

MICROPIPETTE

Principle: Plunger depressed and when released, liquid is drawn into disposable plastic tip.

P2

(0.2–2uL)

P10

(1-10 uL)

P20

(2-20 uL)

P100

(20-100 uL)

P200

(20-200 uL)

P1000

(100-1000 uL)

Filter

serve as a stopper

Gel loading tips

needle-like- thus we need to be very careful when loading

Gel loading tips

used when putting sample in agarose gel or polyacrylamide

Gel loading tips

must not puncture the gel because it can inaccurate the gel, all the specimen will move to the puncture site and will not migrate

PCR CABINET

Also known as “Clean Bench”

PCR CABINET

Similar to biosafety cabinet, but instead of having vertical laminar flow, the air will be coming under and come out horizontally, blown towards the samples

PCR CABINET

• For preparation of PCR Master Mixes

• Product protected from contamination

PCR CABINET

• Air flow: filtered first before going inside the cabinet

• Delivers HEPA-Filtered Air across work surface

- Facilitates worker exposure to materials in use

PCR Machine

considered as the heart of molecular laboratory

PCR Machine

Also known as Thermocycler

PCR Machine

Principle: DNA Replication

PCR Machine

Technically an IN VITRO method of DNA Replication

Reverse Transcriptase

If sample is RNA:

PCR Machine

• One run = ~40 cycles

- Run time: 1 hour 30 mins – 2 hours

• Denaturation

• Annealing

• Extension

Three major cycles of PCR Machine

Denaturation

94 degrees celsius

Annealing

55 degrees celsius

Extension

72 degrees celsius

RT-PCR

used for the detection of RNA (qualitative)

Conventional PCR

used for the detection of DNA (qualitative)

Real-time PCR- aka. Quantitative (Q) PCR

usually used to measure the amount of what is being produced

REFRIGERATOR

Temperature: 2 to 6 degrees Celsius

REFRIGERATOR

For reagents and samples that will be processed immediately

• Storage only until 24 hours or less

ULTRA LOW FREEZER

Also called Minus 80 or Negative 80 Freezer

ULTRA LOW FREEZER

Temperature: -60 to -80 degrees Celsius

- Common is -80 Degrees Celsius

ULTRA LOW FREEZER

For fast freezing of samples

• Best for:

- Eluates

- Storage of specimens

ULTRA LOW FREEZER

What to avoid: Freeze-thaw cycles

SPECTROPHOTOMETER

Principle: Measures light absorbance across the visible and ultraviolet ranges of the spectrum.

SPECTROPHOTOMETER

Used for: Quantification of analytes

SPECTROPHOTOMETER

Asses if the sample is enough or the extracted/isolated DNA

Beer’s Law / Beer Lambert Law

concentration of the solute in the solution is directly proportional in the absorb light and inversely proportional to the transmitted light

High Concentration of Solute

↑ Absorption light, ↓ Transmitted light =

Low Concentration of Solute

↓ Absorption light, ↑ Transmitted light =

VORTEX MIXER

• For mixing of small bottles of liquid

• For suspension or re- suspension of cells

Vortex Mixer

• Principle: transmission of vortex motion

MICROPIPETTES

• Tools used to measure extremely small volumes of liquid

• is accurate to measure a defined range of volume

• Push Button

• Tip Ejector Button

• Tip Ejector Collar

• Volumeter

PARTS OF THE MICROPIPETTE

Push Button

• It is used for aspiration or dispensing the fluid

• It has 2 stops (when pushed or pressed): First stop and Second stop

• It is adjustable by rotating it

First stop

used for aspiration

Second Stop

is used for dispensing the fluid that was aspirated

TIP EJECTOR BUTTON

• It removes the micropipette tip after use. After using the tip, dispose it in a yellow bag.

• If it is infectious, you must dispose it in a solution of sodium hypochlorite.

TIP EJECTOR COLLAR

It will push down the micropipette tip so that it will be removed automatically.

VOLUMETER

• Where the dial windows or numbers are.

Micrometer

used to adjust the numbers shown in the volumeter

P1000

P1000

P1000

P200

P200