Improving Pipetting Techniques

Transcription

1 Improving Pipetting Techniques for Better Accuracy and Performance 49

2 Basic pipetting techniques Pipette cycle Specifications - How to select the optimal volume range for your pipette Basic techniques - Minimizing errors 50

3 Review pipetting cycle - Excludes Tip Insertion Depress 1 kg Tip Ejection 4 kg Hold 1 kg Aspirate 1kg Dispense through Blowout 4 kg Air-Displacement Pipette 51

4 Pipetting techniques Optimizing volume range Micrometer setting Tip immersion angle Tip immersion depth Tip immersion time (macro-volume) Pre-rinsing Aspiration rate Dispensing technique Consistency Hand-warming effects Errors from poor technique can range from 0.1% to 5% or more 52

5 Optimizing volume range Normal Range - 10% - 100% of volume 8Operating at 10% range requires good technique Optimized Range - typically 35% - 100% of volume 35% - 100% 8Less technique dependent 8Assures accuracy and precision Optimizing volume range typically improves accuracy up to 1% 53

6 Setting the micrometer Approach each volume in the same direction each time PRIMIRALY FOR CALIBRATORS ONLY Turn micrometer 1/3 revolution above desired volume Dial-down to volume setting Correctly setting the volume improves accuracy up to 0.2% 54

7 Tip immersion angle Incorrect immersion angle Correct immersion angle Aspirating with pipette perpendicular improves accuracy up to 0.5% (Micro pipettes up to 2.5%) 55

8 Tip immersion depth Pipette size Immersion depth 2 and 10 µl 1 mm 20 and 100 µl 2-3 mm 200 and 1000 µl 3-6 mm 5000 µl and 10 ml 6-10 mm Depth Recommended immersion depths improve accuracy by up to 5% 56

9 Tip immersion time for pipettes Maintain tip immersion time of 1 second (minimum) after aspirating Withdraw tip slowly, smoothly from liquid source Important for large volume samples and viscous liquid samples 57

10 Pre-rinsing pipette tips Pre-rinsing tip with same liquid that is being dispensed Aspirate with tip, and then dispense back into reservoir or to waste Provides identical contact surfaces for all aliquots 58

11 Benefits of pre-rinsing Normalized Volume (%) Number of Pre-Rinses 10μ 200μ 1000μ P-10 P-200 P-1000 Two pre-rinses provides up to 0.2% greater accuracy 59

12 Aspiration rate effects Maintain smooth, controlled aspiration rates Aspiration too quickly causes - Liquid splash-up into shaft Piston and seal damage - Introduction of aerosols / Sample cross-contamination Consistent, controlled aspiration rates improve accuracy 0.2% - 5% 60

13 Dispensing technique Thin-wall tips, such as RAININ FinePoint TM tips, provide maximum droplet dispensing Three techniques 1. Along side-wall 61

14 Dispensing technique Thin-wall tips, such as RAININ FinePoint TM tips, provide maximum droplet dispensing Three techniques 1. Along side-wall 2. Above vessel/ liquid surface 62

15 Dispensing technique Thin-wall tips, such as RAININ FinePoint TM tips, provide maximum droplet dispensing Three techniques 1. Along side-wall 2. Above vessel/ liquid surface 3. Directly into liquid Consistency in pipetting techniques can improve accuracy up to 1% 63

16 Consistency Use consistent - Pipetting rhythm - Pressure on plunger - Speed and smoothness For best consistency - Use an electronic pipette 8 Provides optimum consistency 8 Requires less user technique Correct dispensing technique improves accuracy 0.5% - 1% 64

17 Hand-warming effects Prolonged hand-warming introduces errors of up to 0.5% 65

18 Basic Pipetting techniques Summary Optimizing volume range Micrometer setting Tip immersion angle Tip immersion depth Tip immersion time (macro-volume) Pre-rinsing Aspiration rate Dispensing technique Consistency Hand-warming effects 66

19 Errors in pipetting Error Size Typical Error % Technique Small up to 0.5% Micrometer Setting Pre-rinsing Hand-warming Medium up to 1.0% Optimizing Volume Dispensing Technique Consistency Tip Immersion Angle Large up to 5.0% Tip Immersion Depth Aspiration Rate Note: Small errors usually important to calibration technicians. Micropipette errors may be larger. 67