12: Pipetting Technique
- Page ID
- 123686
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)During the semester, we will perform many experiments that require us to transfer small volumes of liquid with precision and accuracy. Measuring small volumes (10 ml or less) requires that we use special equipment called pipettes. There are two major types of pipettes: serological pipet and micropipet.
Instrument Selection
-
Glass serological pipet
- We will use 5 ml, 10 ml, and 25 ml sizes
- Controlled using a Pipet-Aid = a pump that draws up or dispenses liquid
- The pump may be electronic, scrolling, or bulb
- Micropipet, a.k.a. pipetman
- We will use 2 μl, 20 μl, 100 μl, and 1000 μl sizes
- P2: measures volumes less than 2.00 μL
- P20: measures volumes between 2.0 μL (minimum) and 20.0 μL (maximum)
- P100: measures volumes between 10.0 μL (minimum) and 100.0 μL (maximum)
- P1000: measures volumes between 100.0 μL (minimum) and 1000.0 μL (maximum)
- Draws up and dispenses liquid using a disposable plastic tip
Which pipet should you use?
- Generally, choose the smallest possible instrument to measure the volume you need all at once
- example: if you need 3.5 ml of water, it is best to use a 5 ml serological pipet rather than multiple uses of a P1000
- example: if you need 95 μl of water, it is best to use a 100 μl (P100) pipettor rather than a P1000
- The exception is if you need a precise amount that is slightly more than 1 ml, use a P1000 and pipet in 2 steps.
- example: if you need precisely 1450 μl of water, pipet 750 μl then 700 μl, or some other combination that totals 1450 μl.
- This situation is uncommon, however, and usually you should select the instrument that allows you to measure the entire volume at once.
Use of a Serological Pipet
There are three possible types of Pipete-Aids: electronic, scroll, and bulb (Figure 1). The basic operation is the same, but subtle differences will it easier to use them accurately.
Figure 1. Three different styles of pipet-aids for transferring large volumes. Serological pipets are inserted into the hole on the bottom of each of these pumps. a. Electronic Pipet-Aid b. Bulb-stype Pipet-Aid c. Scroll-style Pipet-Aid
For all Serological Pipette Usage:
- Carefully insert a clean serological pipet into the nose of the Pipet-Aid so that the numbers face you when you hold it.
- Be sure to handle the pipet only near the top, so that your hands will not contaminate the portion that will enter the solution
- Insert the pipet into the solution and draw up the desired amount, measuring at the bottom of the meniscus
- Note that usually the measurement numbers are in reverse order, so if you want to measure 2 ml using a 5 ml pipet, you may need to draw up your liquid to the mark that is labeled 3 ml. Some pipets are marked differently, so pay attention to each one you use.
- Caution: Never allow any liquid to enter the nose of the Pipette-Aid
- Insert the filled pipet into the target container and dispense the liquid
- Carefully remove the used serological pipet from the Pipet-Aid and dispose as instructed by your professor.
Electronic Style Pipet-Aid
- These Pipet-Aids are cordless so you do not need to plug them in or turn them on
- Select the rate of drawing and dispensing by pressing the + and – buttons on the top back of the Pipet-Aid
- Depress the top finger button to aspirate (draw up) liquid and depress the bottom finger button to dispense liquid
Bulb-Style Pipet-Aid
- These Pipet-Aids look like balloons with three valves.
- The largest appendage at the bottom attaches to the pipet while the valves on the other two can be used to control the rate of drawing and dispensing.
- To fill a pipet, start with a deflated bulb (squeeze the bulb and hold the "A" (air) valve at the top)
- Press the "S" (suck) valve on the side to allow air into the bulb, thereby filling the pipet with liquid.
- Press the "E" (expel) valve on the side to dispense the liquid.
Scroll-Style Pipete-Aid
- These Pipet-Aids are tube-shaped with a scroll wheel at the top
- Select the rate of drawing and dispensing by turning the scroll up and down.
- As the scroll moves, the plunger will move up and down. This will pull a vacuum, drawing liquid into the pipet. Scroll down to dispense.
Use of a Micropipetor
At the top of the micropipette is the push button that you will use to manipulate the liquid. Pressing this button will reveal that there are two stops, the first for sucking up the liquid and the second for expelling the liquid. Just below and off to the side is another button used to eject the disposable plastic tips during operation. The black movable ring, or thumbwheel, in the handle allows you to adjust the volume setting of the micropipette. As you turn the thumbwheel, look at the volumeter display. Do NOT turn this wheel past the minimum and maximum volumes for the micropipette to prevent damaging the instrument.
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Figure 2. Diagram of a micropipette.
To determine the volume to which a micropipette is set, you can look at the volume display window on the side of the instrument. See Figure 3 for examples. These values are read vertically and interpreted based on the maximum capacity of the micropipette. In the example below, the left window is from the P20 micropipette and indicates that the instrument is set to measure 6.5 µl.
On the P20 micropipette, notice how the tenths place is shown in red. The center window is from the P100 micropipette and indicates that the instrument is set to measure 92 µL. The right window represents the P1000 micropipette and displays 726 µL. On the P1000 micropipette, notice how the thousands place is shown in red, and the ones place is not displayed.
Figure 3. Volume setting windows for the three most common pipettors used in lab. P20 is set at 6.5µl, P100 is set at 92µl, and P1000 is set at 726µl.
Figure 4. The three most common pipettors used in lab. (a) A 20µl pipettor (P20) can be used to accurately transfer between 2µl and 20µl of a liquid. Here the pipettor is set to 7.5µl. NOTE that the decimal place is red. (b) A 200µl pipettor (P200) can be used to accurately transfer between 20µl and 200µl of a liquid. Here the pipettor is set to 75µl. NOTE that all of the digits are black. (c) A 1000µl (1ml) pipettor (P1000) can be used to accurately transfer between 200µl and 1000µl (1ml) of a liquid. Here the pipettor is set to 750µl. NOTE that the thousands place is red.
NOTE: sometimes in lab we use a P100 and sometimes P200, they both have all black digits in the volume setting
To Set the Volume
Turn the knob on top of the plunger or use the volume adjustment wheel (Figure 2). You will probably need to release the scroll lock on the pipette before you can scroll. Caution: Never try to set pipettor outside its proper range, which is printed on the top of the pipettor!
To Put on the Tips
Gently but firmly press the end of the pipettor onto a pipet tip. P1000s use the large tips; P100s and P20s use the smaller tips; P2s use tiny tips. You should always change tips between samples.
To Suck Up (Aspirate) Liquid
Using your thumb, depress the plunger to the first stop or point of resistance. While holding the plunger down, insert the pipet tip into the solution. Slowly and smoothly release the plunger to draw up the desired amount. Caution: Never allow any liquid to enter the barrel of the pipettor!
To Squirt Out (Expel) Liquid
Insert the filled pipet tip into the target container, just below the surface of any existing liquid, and depress the plunger all the way to the second stop. To prevent bubbles, while holding the plunger down, remove the pipettor and tip from the target container, then release the plunger. Check the pipet tip to make sure you successfully dispensed all the liquid without leaving any drops stuck to the tip.
To Eject the Tip
Point the pipette tip over the waste container. Eject the tip into the waste container by pressing the tip ejector button.