pipetting techniques - eppendorf
TRANSCRIPT
Pipetting Techniques
Best pipetting practice
Forward and reverse pipetting
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1.00
2.00
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10–100 20–200 100–1,000
Mea
sure
men
t er
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[%]
Pipette model [µL]
systematic error random error
998
1,000
1,002
1,004
1,006
1,008
0 1 2 3 4 5 6 7 8 9 10
Vol
ume
[µL
]
Pipetting steps
False pipetting posture: > 45°Correct pipetting posture: vertical
98.4
98.8
99.2
99.6
100.0
100.4
1 2 3 4 5 6 7 8 9 10
Vol
ume
[µL
]
Pipetting steps
1) Correct pipette/tip combinationReduce errors by choosing a pipette/tip combination with the smallest possible air cushion.
2) Correct immersion depth> Immerse as little as possible → preventing liquid transfer on the tip’s outside
> Immerse deeply enough to avoid uptake of air
3) Vertical pipette posture for liquid uptake The hydrostratic pressure changes with the holding angle of the pipette.
4) Prewetting = Saturating of air cushionPrewet tip at least three times to equilibrate air cushion to liquid.
5) Correct liquid discharge> Discharge liquid by touching the wall of the target
vessel → adhesion force of liquid > Volumes below 10 µL: discharge directly into the liquid
6) Slow and continuous working paceTo ensure precise and accurate pipetting results
Volume in μL Depth in mm
0.1–1 1
1–100 2–3
100–1,000 2–4
1,000–10,000 3–6
Transfer Forward pipetting Reverse pipetting
Liquid uptake1. Press operating button down to 1st stop
2. Let operating button move up completely
1. Press operating button down to 2nd stop
2. Let operating button move up completely
Liquid discharge 3. Press operating button via 1st stop down to 2nd stop 3. Press operating button down to 1st stop
Observation 4. No liquid is left in tip after action 4. Liquid is left in tip after action (volume of blow-out)
Small air cushion reduces errors: Setting 100 µL → optimal pipette 10–100 µL
Pipette set to 100 µL: The first and the second pipetting step is insufficient
False pipetting when holding 100–1,000 µL Pipette at an > 45° angle (set volume = 1,000 µL)
1. 2. 3. 4. 1. 2. 3. 4.REST POSITION REST POSITION
1st STOP 1st STOP
2nd STOP 2nd STOP