[1S] UNIT 1.2: Pipetting Techniques

Question 1

____ Pipettes: 100 μl - 1000 μl
____ Pipettes: 10 μl - 100 μl

Answer 1

Blue
White
(Conversion: 1 mL = 1000 μl)

Question 2

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.

Answer 2

F; moves down

Question 3

TYPES OF AUTOMATIC PIPETTES

● Air is displaced by the piston.
● The volume of air displaced is equivalent to the volume of liquid aspirated.

Answer 3

Air Displacement

Question 4

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

Answer 4

Air Displacement

Question 5

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.

Answer 5

Positive Displacement

Question 6

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)

Answer 6

Positive Displacement

Question 7

MODES OF PIPETTING

preferred when dispensing aqueous solutions, which may contain low detergents and protein concentrations

Answer 7

Forward Pipetting

Question 8

MODES OF PIPETTING

usually performed when dispensing viscous or foaming liquids, or when dispensing very minute amount of liquid

Answer 8

Reverse Pipetting

Question 9

Forward Pipetting (Air & Positive)

Answer 9

↓ ↑ ↓ ↑
↓ ↑

Question 10

Reverse Pipetting (Air)

Answer 10

↓ ↑ ↓ ↑
↓ ↑ ↓↑

Question 11

T/F: Pre-rinsing or pre-wetting is necessary

Answer 11

T

Question 12

For greater uniformity and precision of dispensing

Answer 12

Pre-rinsing / Pre-wetting

Question 13

1. How to pre-rinse/wet?
2. When should we pre-rinse/wet?

Answer 13

1. 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
2. Every time you change a tip & Every time you increase the volume setting

Question 14

IS WIPING NECESSARY?

T/F: Necessary only when pipetting viscous liquids such as cream.

Answer 14

T - Wiping the outside of the tip or the capillary with a clean tissue (careful not to touch the orifice).

Question 15

IS WIPING NECESSARY?

Choice of tissue

Answer 15

Resistant
Lint-free
Inert to acids & solvents

Question 16

IS WIPING NECESSARY?

T/F: Wipe the tip when you are working with high risk/infectious specimens.

Answer 16

F; Never wipe

Question 17

IS WIPING NECESSARY?

Fluid depth penetration is only ______.

Answer 17

< 3mm

Question 18

RECOMMENDATIONS FOR PIPETTING DIFFERENT SOLUTIONS: PIPETTE, TIP, TECHNIQUE

• Pipette: Air displacement
• Tip: Standard
• Technique: Forward
• Examples: Buffers, diluted salt solutions

Answer 18

Aqueous solution

Question 19

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

Answer 19

Viscous solution

Question 20

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

Answer 20

Volatile compound

Question 21

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

Answer 21

Body Fluids

Question 22

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

Answer 22

Nucleotide solutions

Question 23

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

Answer 23

Radioactive compounds

Question 24

RECOMMENDATIONS FOR PIPETTING DIFFERENT SOLUTIONS: PIPETTE, TIP, TECHNIQUE

• Pipette: Air displacement
• Tip: Filter
• Technique: Forward
• H2SO4 , HCl, NaOH

Answer 24

Acids/ Alkalis

Question 25

RECOMMENDATIONS FOR PIPETTING DIFFERENT SOLUTIONS: PIPETTE, TIP, TECHNIQUE

• Pipette: Air displacement, Positive displacement
• Tip: Filter, Positive displacement
• Technique: Forward or reverse

Answer 25

Toxic Samples

Question 26

WHAT TECHNIQUE/MODE TO USE WHEN PIPETTING WHOLE BLOOD?

Answer 26

↓ ↑ ↓ ↑ ↓ ↑
↓ ↑

Question 27

Basic Workflow in Molecular Biology Laboratory

Answer 27

Sample Collection → Nucleic Acid Extraction → Nuclein Acid Amplification → Nucleic Acid Detection