1 Pipetting Exercise & Reagent Procedures

• Pipette is a laboratory tool commonly used in chemistry, biology and medicine to transport a measured volume of liquid, often as a media dispenser.
• Pipettes are usually seen in micropipette form, which can only measure up to 1000 uL.
• Glass/serological pipette are mostly used if there is a faulty mechanism in automatic pipet or if we need to reconstitute the quality control reagents
• Micropipette is used to measure and dispense small volumes of liquid; mostly used in molecular biology and diagnostics laboratory
• Most common micropipettors are:

  • P20 → which measure volumes from 2.0-20.0 uL
  • P200 → which measure volumes from 20.0-200.0 uL
  • P1000 → which measure volumes from 200.0- 1000.0 uL
• Parts of the micropipette:

  • Plunger button – creates the vacuum
  • Tip ejector button - releases the disposal tips
  • Volume adjustment knob
  • Digital Volume indicator
  • Plastic shaft – supports the entire pipet
  • Tip ejector arm – connected to tip ejector button, which helps to force the full release of the tips.
  • Disposable tips
• In pipetting procedure, always observe the appropriate immersion depth of 1-3mm ► immersing the tip too deeply or not deep enough will affect the selected volume.
• In pipetting procedure, too shallow will cause the introduction of air. When air is introduced, it will cause a false measurement of volume.
• The horizontal line in a micropipette denotes a decimal point.
• Air displacement pipette has a general use for aqueous products and is most commonly used in molecular biology lab; uses pipet tips
• Positive displacement pipette is used for highly viscous and volatile liquids; uses the tip itself of the pipet → piston
• Single channel pipette (fixed or variable) has a fixed or adjustable volume → P10, P100, P1000; its has only one orifice
• Multichannel pipette is commonly used in microplate applications and has 4-8 orifice; it saves time and has lesser prone to contamination
• Electronic pipette is used for repetitive pipetting; it removes clerical and technical errors
• Serological pipette can dispence over 1ml of volume; it uses up to date in reconstitution of quality control reagent
• Bulk reagent dispenser dispenses from the reagent bottle; normally used if we are dispensing fluids from the same container
• Automated liquid handling system is used for bulk manufacturing
• Standard Tip is a multipurpose tip; commonly used in clinical laboratory but not in molecular lab because:

  • it is unsterile
  • contaminated -
  • has nucleases
  • no filter
• Sterile Filter Tip prevents cross-contamination; it is the most commonly used in molecular bio applications
• Extended length pipette tips are used to easily reach bottom of tubes
• Low retention tip reduces sample loss
• Wide orifice tips is used for fragile cellular samples (to avoid damaging the cellular sample; normally used for cell cultures and cytology)
• Gel loading tips is used for loading to agarose or acrylamide gels
• Solvent safe carbon filtered tips is used for strong acids or bases (has dark filter bec. of the carbon)
• Individually wrapped tips is used for strict aseptic purposes
• Forward pipetting is a technique used for pipetting and mixing sample or reagent to another liquid; it is normally used for regular aqueous sample such as urine, CSF, ascitic fluids, etc.
• Reverse pipetting is used for high viscosity solutions (or those that foam)
• Repeat pipetting is used for dispensing into microplates or tubes
• The first and second directions in repeat pipetting are called pre-wetting technique.
• In repeat pipetting, dispensing the first stop, and second stop is called pipette mixing
• Pipetting accuracy is mostly dependent on user’s skill level and techniques; it should be emphasized on the lab’s quality assurance
• In storing pipettes when not in use, the pipette should be vertical, never store the pipettes horizontally because fluids may tend to accumulate in the orifice
• Ergonomics is the handling of certain instruments or machines while putting into mind the health of person and to minimize the risk of injury
• Sterility is very important in molecular laboratories (because the main contaminant is nucleic acids)
• Buffer is a solution which tends to maintain a constant pH when excess acid or base is added.
• Concentrated solution for some commonly used acids and bases, the maximum solubility (at room temperature) in an aqueous solution or as a pure liquid
• Concentration is the relative amount of solute and solvent in a solution
• Hydrates are compounds containing water chemically combined in a definite ratio.

  • Computations using formula weight must take the water molecules into account.
• Miscible is the ability of two liquids to be completely soluble in one another
• Molality is a concentration unit (m); defined as the number of moles of solute divided by the number of kilograms of solvent.