Name: Choong Mel June
Group: RBS2 – Group 1
Subject: BABS 2223 Principles of Biotechnology
Date: 15 May 2014

Title:
Basic Laboratory Techniques – Part I: Basic Laboratory Techniques: Pipetting

Objectives: 1. To learn which pipettes are used for desired volumes. 2. To learn correct procedures for measurement and transfer of liquids. 3. To become familiar with the appearance of certain volumes in pipet tips.

Introduction: Micropipettes are equipment used to transfer and measure very small amounts of liquid as low as 0.003 ounce (0.1 millilitres) in laboratory. Standard micropipettes used in laboratory settings feature a few basic parts including an adjustment dial and a plunger button. Normally, micropipettes are consisting several of components, such as volume adjustment dial and a digital volume indicator. These pipettes also get used to different types of disposable tips at the end of a shaft which is one part of special of micropipettes. Liquids are drawn into the disposable plastic tips fitted at the end of micropipette shafts instead of into the shafts themselves. There are 2 main buttons found on micropipettes, which are the plunger button and the tip ejector button. The pipette will immediately release the disposable tip at the end of the device by pushing the ejector button. Most of plungers of micropipette are designed to stop at two different positions. Pipette will draw the desired volume of liquid up into its disposable tip by depressing the plunger to the first stopping position and slowly releasing it. A micropipette's plunger is then pressing down further to the second stopping point to release the volume of fluid drawn into at the first stop. For accuracy measurement, pipette plungers should be pressed down to first stop point before the pipette tip gets inserted into a sample. Volume adjustment dial of a micropipette is to help to ensure accurate measurement. It functions as the adjuster by adjusting the volume of liquid entering the pipette's tip. As this dial is adjusted, the digital readout on the volume indicator will change to reflect the adjustment. Several different sizes, types and styles of micropipettes are available. The most common micropipette sizes have 3; they are including P20, P200, and P1000. Each different size of micropipettes is designed to measure different volume ranges of liquids. P20 micropipettes are used to measure volumes within the range of 0.02 and 0.7 ounce (0.5 and 20 millilitres). Meanwhile P200 micropipettes are used to measure volumes between 0.7 and 6.8 ounces (20 and 200 millilitres). P1000 micropipettes are used to measure liquids with a volume in the range of between 3.4 and 33.8 ounces (100 and 1,000 millilitres) which may considered as the larger micropipettes available. There are two common types of micropipette, which are air displacement pipettes and positive displacement pipettes.

[pic][pic] Air displacement pipettes Positive displacement pipettes

A few things to know about using the micropipettes: 1. Never adjust the volume beyond the range of the micropipette. Micropipette should not be adjusted below 0 µL. P20 micropipette should be adjusted in the range of 0 - 20 µL; P200 micropipette should be adjusted in the range of 0 - 200 µL and P1000 micropipette should be adjusted in the range of 0 - 1000 µL (over 1 mL). 2. Never force the volume adjustor dial. If the knob becomes difficult to adjust it probably means that you are exceeding the limits for the pipette or the pipette is damaged. 3. Do not drop the micropipette. 4. Always use a smooth motion when using the micropipette. This will help give accurate measurements and also prevent breakage of the pipette. There should not be “snapping” noises. 5. Always keep pipettes upright. 6. Never lay a pipette with liquid in the tip on the bench. 7. Always choose the appropriate size pipette for the volume you are measuring. 8. Always dispose of tips in the appropriate waste receptacle.

Methodology:
1. The tip was fitted to the end of the shaft.
2. Then, the tip was pressed down and twisted slightly to ensure an airtight seal.
3. The pipette was hold in a vertical position.
4. Then, the plunger was depressed to the first stop. Air equal to the volume of the setting was displaced.
5. The tip was then immersed into the liquid.
6. After that, the plunger was released back to the rest position. A second was waited for liquid to be sucked up into the tip. The volume of liquid in the tip was equal to the volume of the setting of the micropipette.
7. The tip was placed at an angle (10° to 45°) against the wall of the vessel receiving the liquid.
8. After that, the plunger was depressed to the first stop and waited for one second.
9. Then, the plunger was pressed to the second stop to expel all the liquid.
10. The end of tip was moved away from the liquid and the plunger was released to the rest position.

Results:
|Volume |Pipette No. |Weight 1 |Weight 2 |Weight 3 |Mean |S.D. |
|12 µl |SL 20 |0.013g |0.016g |0.014g |0.0143g |0.001528 |
|157 µl |SL 200 |0.1568g |0.16g |0.157g |0.1577g |0.001815 |
|645 µl |SL 1000 |0.647g |0.638g |0.649g |0.6447g |0.005860 |

Discussion: Micropipettes are the standard laboratory equipment used to measure and transfer small volumes of liquids. Different design of micropipettes with differing levels of accuracy and precision, from single piece glass pipettes to more complex adjustable or electronic pipettes. Pipettes can be grouped into two categories: common pipettes and specialized pipettes. For example, common pipettes are air displacement micropipettes, positive displacement pipettes, volumetric pipettes, graduated pipettes, Pasteur pipettes and transfer pipettes. Meanwhile for specialized pipettes, there are included pipetting syringe, Van Slyke Pipette, Ostwald-Folin Pipette, glass micropipette, micro fluidic pipette and extremely low volume pipettes.

Typically, micropipettes in this laboratory come in 3 different sizes each of which measures a different range of volumes. They are P20, P200 and P1000. These sizes are noted on the top of the plunger button.
|Size Micropipette |Range of volumes measured |
|P20 |0.5-20µl |
|P200 |20-200µl |
|P1000 |100-1000µl |

The pipette terms of adjustment is altering the pipette so that the dispensed volume is within the specifications. The black volume adjustment dial near the top of the micropipette allows us to adjust the volume that is measured. By dialling it to the left or right is to increase or decrease the volume. The digital readout shows the volume that will be measured. As the volume adjustment dial is turned on, the numbers in the digital readout will be changed. On each of the three sizes of micropipettes (P20, P200, P1000) the digital readout has three numbers. These three numbers correspond to different volumes on the different size pipettes. The figure below shows the instructions on interpreting digital readout.

[pic] - In a P100, the top number refers to 1000’s of µl, the middle number refers to 100’sµl and the bottom number refers to 10’s of µl’s. - In a P200, the top number refers to 100’s of µl, the middle number refers to 10’s µl and the bottom number refers to µ’ls. - In a P20, the top number refers to 10’s of µl, the middle number refers to µl’s and the bottom number refers to 1/10ths of µl.

There are many types of pipettes. One of them is air displacement pipettes (Pipetman models: single and multi-chanel). They are recommended for aqueous samples and for general laboratory work. Besides, they used for standard pipetting application which is highly accurate. Air displacement pipettes, often referred to as pipettes, and are generally used for smaller volumes of liquid, from 1 ml to as little as 0.1 ul (0.0001 ml). To avoid micropipettes contact with liquid, they are always used with disposable tips. The pipette contains a piston in a cylinder or a capillary tube that moves to an appropriate position when the volume is set. When an operating button is depressed the piston moves so that the set volume of air is expelled from the pipette. The disposable tip is placed in a liquid and the button is released, creating a vacuum that pulls the set volume into the tip. The force of the liquid will be out by depressing the button again. With displacement pipettes, there is always a specified volume of air between the pipette piston and the liquid (cushion of air). The piston is a permanent part of the pipette. But, conditions such as temperature, atmospheric pressure, gravity and viscosity of the solution may have an effect on the performance of air displacement pipettes.

[pic]

When the push-button of air displacement pipette is pressed on, the piston inside the instrument moves down to let the air out. Air is displaced by the piston. The volume of air displaced is equivalent to the volume of liquid aspirated. The schematic drawings below show how the piston determines the volume of air displaced and subsequently the volume of sample aspirated. From the working principle of air displacement pipettes, we able to understand the pipette terms aspirate is to draw up the sample and dispense is to deliver the sample.

[pic] [pic]
[pic] [pic]

All air-displacement pipettes are not the same. For applications such as DNA sequencing and biochemical procedures that require tiny, valuable, samples, two Pipetman models provide exceptional accuracy and precision, from 0.1 to 2 µl for Model P2 and 0.5 to 10 µl for Model P10. Minimum air space between the piston and sample makes performance less sensitive to variations in temperature and liquid properties such as vapour pressure and density. Select a pipette with a protected piston, so there is no risk of sample contact or cross-contamination. Grease-free Pipetman requires less maintenance, reduces the risk of leaks and contamination. Unlike most pipettes with greased pistons, Pipetman uses “dry seal” technology. To prevent leaking occurs, greased pistons must be regreased regularly. Another cause of leaks is the grease may be altered by vapours from liquids pipette. Grease can also introduce contaminating particles into the body of the pipette.

There are some factors affecting the accuracy of air displacement pipettes. First is the hydrostatic pressure induced errors. With an air-displacement pipette, the piston is displaced to produce a pressure drop inside the tip touching the liquid. The tip will be filling by the atmospheric pressure due to the inside pressure together with the hydrostatic pressure induced by the liquid column equal to the ambient pressure. Because of the hydrostatic pressure is directly proportional to the height of the liquid column, so there is a volume difference between the displaced air volume and the liquid volume. Hence, the air volume displaced by the piston is bigger than the volume of the liquid in the tip. Next, the factor affecting the accuracy of air displacement pipettes is the tip of the pipette. For a typical conically shaped tip the liquid height h versus the liquid volume is not linear. This induces non-linear calibration, which is difficult to correct at least with a mechanical pipette. Moreover, if a differently shaped tip is used where the height versus the volume behaves unlike in the tip used for calibration, the calibration is not. It is not only the shape of the tip that is important but also the nominal volume of the tip. In general, one should always use the smallest pipette with proper tip available for each volume pipetted, because it can minimize the percentage error after calibration. On the other way, pipetting angle also is one of the factors affecting the accuracy of air displacement pipettes. Calibration is performed by keeping the pipette vertically oriented. In routine work it is common to tilt the pipette during aspiration. This causes a volume greater than the volume set of liquid to enter into the tip because of the reduced effect of hydrostatic pressure. In practice, the angle does not stay the same during pipetting when tilted, and the difference in accuracy can be even more.
[pic]
Altitude is categorized as the factor affecting the accuracy of air displacement pipettes too. If the pipettes are calibrated at normal temperature and pressure (NTP) close to sea level, but used at areas of high altitude, less liquid is aspirated. This is because air-volume displacement error is inversely proportional to the atmospheric pressure
P0, being lower at high altitudes. It is recommended to use higher settings to compensate the error caused by low atmospheric pressure at a high altitude when re-calibration is not able to do.
[pic]
The next factor affecting the accuracy of air displacement pipettes is liquid density. High density of the liquid is which is higher than that of purified water, smaller the volume entered into the tip is. The error is usually significant. However, it is evident that the change in density due to temperature does not produce a large error. By the way, if the liquid is very viscous, volatile or has a high surface tension, then the pipetting technique used will be the one to determine the accuracy.
[pic]
Furthermore, temperature affects the pipetting results too, especially when using air displacement pipettes. This is illustrated by the following Formula 2 derived for a typical two-compartment model and applying gas laws on it.
Formula 2DV = (1-Tp / Tt ) V , where
DV = difference in piston displaced air volume and liquid volume in the tip
Tp = absolute temperature of the gas inside the pipette (Kelvins)
Tt = absolute temperature of the gas inside the tip (Kelvins)
V = set value of the aspirated liquid volume This is due to the error is proportional to the absolute temperature. Moreover, the volume set, time elapsed and the design of the pipette has an impact on the error induced. When work with cold liquids, we are most recommended to do not pre-rinse the tip and change the tip between every pipetting. This is because pre-rinsing decreases the air temperature inside the tip (when stored at room temperature) causing the volume to drop by according to the gas laws of physics. This can be explained by this way, the more often the same tips is used, the colder the air gets and the smaller the aspirated volume until minimum level is achieved. Besides, if the tip is stored in a fridge, we need to heat up it to the same level of temperature as the pipette before using it.

[pic]

Another type of pipettes is positive displacement pipettes (Microman models and Distriman models) which are less commonly used. They are similar to air displacement pipettes and are used to avoid contamination and for volatile or viscous substances at small volumes. They used for applications like PCR and other DNA amplification techniques and recommended for problem samples (viscous, dense, volatile, radioactive and corrosive). The micro-syringe tips used in positive displacement pipettes are disposable. Besides, they direct contact of the piston with the sample (no air cushion). The major difference is that the disposable tip is a micro syringe (plastic), composed of a plunger which directly displaces the liquid.
[pic]

Positive-displacement pipettes work like a syringe. As written on above, no air cushion between the disposable piston and the sample to expand or contract, the aspiration force remains constant, unaffected by the physical properties of the sample. This allows the Microman operator to pipette very viscous or high density samples, such as mercury or toothpaste.
[pic] [pic]
[pic] [pic] With Microman pipettes, the piston comes into direct contact (positive-displacement) with the sample. There is no air cushion to expand or contract in response to the density or the temperature of samples. The desired volume is aspirated completely.
The piston seal prevents biohazard, radioactive or corrosive aerosols from entering the instrument. During dispensing, the piston wipes the internal capillary wall, assuring accurate dispensing of almost any viscous sample, from glycerol to glue. To prevent contamination, the capillary and piston may be changed after each sample. So, they are ejected automatically.

The forward mode is the usual way of pipetting with an air-displacement pipette like Pipetman. The forward mode for positive-displacement pipettes like Microman eliminates the purge stroke. Meanwhile, the reverse mode is only possible with air-displacement pipettes. It is used for solvents or slightly viscous liquids. For forward pipetting technique, it is used for standard solutions including buffers, water, dilute saline, and dilute acids and bases. Besides, it is more accurate and precise results than reverse pipetting technique. Entire volume of liquid aspirated into the pipette tip is dispensed by depressing the push button to the first stop. Moreover, the tip is placing 2-5 mm below the surface of the liquid and waiting for about 1 – 2 seconds, then, the solution is drawing slowly up when using forward pipetting technique in laboratory. When withdrawing the liquid, the tip must touch it against the sides of the reservoir to remove excess liquid. Then, the liquid sample is delivering by depressing push button to the first stop, and then blowing out the remaining liquid by depressing to second stop. This action is to empty the tip. For reverse pipetting technique, it is used for slightly viscous solutions, small volumes, buffers with detergent or protein, and solutions that foam easily. In reverse pipetting technique, the push button is depressing all the way to the second stop which is differing with forward pipetting technique. And, the tip is placing 2-5 mm below the surface of the liquid and waiting for about 1 – 2 seconds, then, the solution is drawing slowly up. Same to forward pipetting technique, when withdrawing the liquid, the tip must touch it against the sides of the reservoir to remove excess liquid. To deliver the solution, the push button is depressing to the first stop. A portion of the solution will remain in the pipette tip and this volume should not be delivered.
[pic] [pic]
Forward pipetting technique Reverse pipetting technique
Questions:
1. If a 20 – 200 µl micropipette is set to 0 how many µl is it set to measure?

How many mL is this?

- 43 µl is set to measure and it is 0.043mL.

2. Why should you avoid touching the micropipette tips? - We should avoid touching the micropipette tips because it may causes contamination. Besides, if we handle the tips, the heat transferred from our hands to the tips can affect deliver volumes.

3. Why should you avoid submerging the micropipette tip too deep in the liquid? - We should avoid submerging the micropipette tip too deep in the liquid due to too much immersion will cause liquid stick to the outside of tip and more liquid will deliver than was desired. Moreover, too little immersion will result in air bubbles and cause inaccuracy. Large volume pipettes (1-5mL) should be immersed 5-6 mm below the meniscus. Smaller volume pipettes should be immersed 2-3 mm below the meniscus.

4. What happens if you push the plunger to the second stop before drawing up the liquid? - Micropipettes have a 2-position plunger: the first stop is for pulling up the correct volume, and the second stop is to blow out any additional solution that remains in the tip due to capillary action. If we push the plunger down to the second stop before drawing up the liquid, we are not pulling up the right volume.

5. What does the phrase – pipetting up and down mean and how is this technique used? - D

6. On what part of a microcentrifuge tube should you write a label? - D

7. Describe why step 4 is needed in the experiment. - D

Conclusion: