exp 1 - pipetting_complete.docx

7/28/2019 Exp 1 - Pipetting_complete.docx

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Title :Pipetting

Objectives1. To practice using micropipette properly by moving water from a flask to a

microcentrifuge tubes

Methods

Rules to prevent damaging the micropipettes

1. The micropipette are never used or the volume of adjuster beyond are rotatedbeyond its maximum or minimum range

2. The micropipette are never used without a clean tip in place3. The filled micropipettes are always hold vertically and never have been lay down.

4. Any part of the micropipette are not allowed to be immersed in any fluid5. The plunger are not allowed to snap back after withdrawing or ejecting a sample6. The micropipette tip are never been flamed

How to use a micropipette

1. The volume adjuster is rotated to desired setting2. A proper-sized tip are firmly sat on the end of the micropipette3. The tube is held firmly between the thumb and forefinger during withdrawing or

expelling fluid.4. The microcentrifuge tube is held nearly to the eye level to observe the change in

the fluid level in the pipette tip. The tube in the tube rack was not pipetted.5. For a better control, the micropipettor are grasped in the palm and the fingers werewrapped around the barrel. The piston was working with the thumb. Themicropipettor is held vertically when filling it.

6. Most of the micropipettor have two positions with friction stop. The depressed firststop measures the desired volume. The depressed second stop introduces anadditional volume of air to blow out any solution remaining in the tip. These frictionstops are noticed. It is felt with the thumb.

To withdraw the sample from a microcentrifuge tube

1. First, the correct micropipette for a volume desired are checked2. The desired volume is dialled3. The end of the micropipette is gently pressed into a sterile pipette tip firmly4. The plunger is depressed to the first stop and it is held in this position5. The tip is dipped into the solution to be pipetted and the fluid is drawn into the tip

gradually by releasing the plunger. The tip remains in the solution while releasingthe plunger.


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6. The pipette tip are slide out along the inside wall of the reagent tube to dislodgeany excess droplets adhering to the outside of the tip

7. It is checked, so that there is no any air space at the very end of the tip.8. If there is any air space at the end of the tip or air bubbles within the sample in the

tip, it is then carefully expelled back the sample into its microcentrifuge

To expel the sample into a reaction tube

1. The tip of the pipette touches to the inside of the reaction tube into which thesamples will be emptied.

2. The plunger is slowly depressed to the first stop to expel the sample. The secondstop is depressed to blow out the last bit of the fluid. The plunger are held in thedepressed position and the tip is removed from the solution while dragging the tipacross the wall of the tube.

A. Quantitative estimation of pipetting accuracy

1. 4 micro centrifuge tubes are obtained. Each one is labelled according to the Datatable.

2. A micro centrifuge tube are weighed in the fine balance and the balance are zero3. The instruction on How to use a micropipette are followed and specific amount of

water are add on to the micro centrifuge tube. The amount of water to be added isreferred to the Data table one. The microcentrifuge tube is placed on the finebalance to determine the weight of the water. The weight is recorded in the Datatable 1. The centrifuge are emptied. The weight of each volume for three times aremeasured and recorded.

4. The percentage error, average error and the standard deviation are completed in

the data table 1.

Results

Micropipette Volume Expectedweight

ActualWeight

Average % Error StandardDeviation


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P10 8uL 0.008g 0.008g0.008g

0.00 %08uL 0.008g 0.008g 0.00 %

8uL 0.008g 0.008g 0.00 %

P100 40uL 0.04g 0.043g0.041g

7.50 %1.6 x 10-340uL 0.04g 0.041g 2.50 %

40uL 0.04g 0.040g 0.00 %P100 68uL 0.068g 0.068g

0.069g0.00 %

1.0 x 10-368uL 0.068g 0.069g 1.47 %

68uL 0.068g 0.070g 2.94 %

P1000 550uL 0.55g 0.552g0.550g

0.36 %2.1 x 10-3550uL 0.55g 0.549g 0.18 %

550uL 0.55g 0.548g 0.36 %

P1000 1000uL 1g 0.998g0.9977g

0.20 %2.9 x 10-31000uL 1g 0.998g 0.20 %

1000uL 1g 0.997g 0.30 %

Calculation Table for Standard Deviation

Discusion

P10

x x-u (x-u)

0.008g 0 0

0.008g 0 0

0.008g 0 0

(x-u) 0

P100

x x-u (x-u)

0.043g 0.002 4.0 x 10-6

0.041g 0 0

0.040g -0.001 1.0 x 10-6

(x-u) 5.0 x 10-6

P100

x x-u (x-u)

0.068g -0.001 1.0 x 10-6

0.069g 0 00.070g 0.001 1.0 x 10-

6

(x-u) 2.0 x 10-6

P1000

x x-u (x-u)0.552g 0.002 4.0 x 10-

6

0.549g -0.001 1.0 x 10-6

0.548g -0.002 4.0 x 10-6

(x-u) 9.0 x 10-6

P1000

x x-u (x-u)

0.998g -0.002 4.0 x 10-

0.998g -0.002 4.0 x 10-

0.997g -0.003 9.0 x 10-

(x-u) 17.0 x10-6


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The accuracy of micropipette depends on the percentage of error. The lower thepercentage or error, the higher the accuracy of the micropippete. From our experiment,P10 micropipette is more accurate which shows the most lowest number of percentageerror which is 0%. Besides that, by calculating the standard deviation, the most precisemicropipette can be identified. The smaller the standard deviation, the more precise the

micropipette is. Therefore, from the value of standard deviation calculated above, themost precise micropipette is P10.

Conclusion

From the analysis, the most sensitive and precise micropipette is P10

exp 1 - pipetting_complete.docx

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