[1S] UNIT 1.2: Pipetting Techniques Flashcards
____ Pipettes: 100 μl - 1000 μl
____ Pipettes: 10 μl - 100 μl
Blue
White
(Conversion: 1 mL = 1000 μl)
TYPES OF AUTOMATIC PIPETTES
T/F: When the push-button is pressed on an air-displacement pipette, the piston inside the instrument moves up to let air out.
F; moves down
TYPES OF AUTOMATIC PIPETTES
● Air is displaced by the piston.
● The volume of air displaced is equivalent to the volume of liquid aspirated.
Air Displacement
TYPES OF AUTOMATIC PIPETTES
● Recommended for aqueous samples and for general laboratory work
● Always have a cushion of air (dead volume) between the pipette piston and the liquid sample
● The piston is a permanent part of the pipette
Air Displacement
TYPES OF AUTOMATIC PIPETTES
● Works like a syringe
● With no elastic air cushion to expand or contract, the aspiration force remains constant, unaffected by the physical properties of the sample.
Positive Displacement
TYPES OF AUTOMATIC PIPETTES
● Recommended for problem samples (viscous, dense, volatile, radioactive, corrosive)
● Direct contact of the piston with the sample (no air cushion)
● Disposable piston (not a permanent part of the pipette)
Positive Displacement
MODES OF PIPETTING
preferred when dispensing aqueous solutions, which may contain low detergents and protein concentrations
Forward Pipetting
MODES OF PIPETTING
usually performed when dispensing viscous or foaming liquids, or when dispensing very minute amount of liquid
Reverse Pipetting
Forward Pipetting (Air & Positive)
↓ ↑ ↓ ↑
↓ ↑
Reverse Pipetting (Air)
↓ ↑ ↓ ↑
↓ ↑ ↓↑
T/F: Pre-rinsing or pre-wetting is necessary
T
For greater uniformity and precision of dispensing
Pre-rinsing / Pre-wetting
- How to pre-rinse/wet?
- When should we pre-rinse/wet?
- Aspirate the liquid to be dispensed with the tip, and then dispense back into the original reservoir or to waste. May be done 2 to 3 times
- Every time you change a tip & Every time you increase the volume setting
IS WIPING NECESSARY?
T/F: Necessary only when pipetting viscous liquids such as cream.
T - Wiping the outside of the tip or the capillary with a clean tissue (careful not to touch the orifice).
IS WIPING NECESSARY?
Choice of tissue
Resistant
Lint-free
Inert to acids & solvents
IS WIPING NECESSARY?
T/F: Wipe the tip when you are working with high risk/infectious specimens.
F; Never wipe
IS WIPING NECESSARY?
Fluid depth penetration is only ______.
< 3mm
RECOMMENDATIONS FOR PIPETTING DIFFERENT SOLUTIONS: PIPETTE, TIP, TECHNIQUE
- Pipette: Air displacement
- Tip: Standard
- Technique: Forward
- Examples: Buffers, diluted salt solutions
Aqueous solution
RECOMMENDATIONS FOR PIPETTING DIFFERENT SOLUTIONS: PIPETTE, TIP, TECHNIQUE
- Pipette: Air displacement, Positive displacement
- Tip: Standard or wide orifice
- Technique: Reverse
- Comment: Pipette slowly to avoid bubble formation
- Examples: Protein and nucleic acid solutions, glycerol, Tween 20/40/60/9
Viscous solution
RECOMMENDATIONS FOR PIPETTING DIFFERENT SOLUTIONS: PIPETTE, TIP, TECHNIQUE
- Pipette: Air displacement, Positive displacement
- Tip: Filter, Positive displacement
- Technique: Reverse
- Comment: Pipette rapidly to avoid evaporation. Carbon filter tips prevent vapor going into the pipette
- Examples: Methanol, Hexane
Volatile compound
RECOMMENDATIONS FOR PIPETTING DIFFERENT SOLUTIONS: PIPETTE, TIP, TECHNIQUE
- Pipette: Air displacement
- Tip: Standard or wide orifice
- Technique: Pipetting of heterogeneous samples
- Residual liquid can be found on the outer surface of the tip. Wipe the tip against the edge of the vessel to remove this liquid before dispensing
- Examples: Whole blood, serum
Body Fluids
RECOMMENDATIONS FOR PIPETTING DIFFERENT SOLUTIONS: PIPETTE, TIP, TECHNIQUE
- Pipette: Air displacement, Positive displacement
- Tip: Filter or wide orifice, Positive displacement
- Technique: Forward
- For genomic DNA wide orifice tips can be used to eliminate mechanical shearing
- Examples: Genomic DNA, 3 H-thymidine
Nucleotide solutions
RECOMMENDATIONS FOR PIPETTING DIFFERENT SOLUTIONS: PIPETTE, TIP, TECHNIQUE
- Pipette: Air displacement, Positive displacement
- Tip: Filter, Positive displacement
- Technique: Forward
- Examples: 14 Carbonate, 3 H-thymidine
Radioactive compounds
RECOMMENDATIONS FOR PIPETTING DIFFERENT SOLUTIONS: PIPETTE, TIP, TECHNIQUE
- Pipette: Air displacement
- Tip: Filter
- Technique: Forward
- H2SO4 , HCl, NaOH
Acids/ Alkalis
RECOMMENDATIONS FOR PIPETTING DIFFERENT SOLUTIONS: PIPETTE, TIP, TECHNIQUE
- Pipette: Air displacement, Positive displacement
- Tip: Filter, Positive displacement
- Technique: Forward or reverse
Toxic Samples
WHAT TECHNIQUE/MODE TO USE WHEN PIPETTING WHOLE BLOOD?
↓ ↑ ↓ ↑ ↓ ↑
↓ ↑
Basic Workflow in Molecular Biology Laboratory
Sample Collection → Nucleic Acid Extraction → Nuclein Acid Amplification → Nucleic Acid Detection