Now that you have practiced calculations and conversions, you are ready to become familiar with some of the essential tools of the biotechnician. In the next few labs, you will learn to use the micropipetter, the balance and the centrifuge. These three tools are used daily in many bioscience labs around the world.
Your performance will be satisfactory when you are able to follow these Good Laboratory Practices (GLPs) and make them a habit for every lab:
Keep your work area clear of unnecessary items
Keep everything you need within reach
Gather all materials before you begin working
Set up disposal areas before you begin working
Label each container BEFORE you fill it
Change gloves often to avoid contamination
Never wear your gloves out of the lab
Never do protocols from memory; always read every step every time you perform a procedure, and then check it off as it is completed
Always cap bottles of stock solutions and chemicals when finished
Never hold a solution in a micropipetter; always eject immediately
Materials (per group)
20-50 mL sugar solution (dyed any color)
20-50 mL each of distilled water and water dyed blue
small beakers (4)
1.5 mL microcentrifuge tubes (12 per group member)
microcentrifuge tube rack
set of 3 micropipetters
box of 20-200 µL tips
box of 100 – 1000 µL tips
wash bottle with 70% ethanol
A. Organizing Your Work Space
When your work requires aseptic (sterile) conditions, you should wash the benchtop with 70% ethanol. Although this procedure does not need to be sterile, wash the table with ethanol to get into the habit. Collect everything (including paper towels) you will need for the lab, except things like the stock solution bottle that will be shared by the whole class. Each person in your group will do each of the measurements, so make sure you have enough containers. In order to work efficiently, you should arrange everything at your workstation so that you can reach it easily. The center of the workstation should be clear of items you are not immediately using. Always have a waste beaker for used tips when you are micropipetting; do not use the sink for disposal of tips!
B. Micropipetting Practice
GLP Tip: Never lay a micropipetter down with a filled tip or hold it upside down or sideways. The liquid will not leak out if you hold it upright but it may enter the instrument if you hold it upside down, and contamination will result.
Practice setting the volume on the micropipetters; each person in your group should set at least one and have it checked by other group members and/or your instructor. Look at the top of the micropipetter to identify its measuring range. Remember that the highest value listed on the top is the largest volume you can measure on that pipet. On a 100 to 1000 µL micropipetter, the largest measurable volume is 1000 µL; on a 20-200 micropipetter, it is 200 µL. Likewise, the smaller value in the range is the smallest measurable volume; on a 2-20 µL micropipetter, the smallest measurable volume is 2 µL. Set a 100-1000 µL pipetter to 0.45 mL, a 20-200 µL pipetter to 0.15 mL, and a 2-20 µL to 0.015 mL. What are these values in µL? You should practice doing that kind of conversion in your head; it will be useful when working in a lab.
Have a graduated 1.5 mL microcentrifuge tube in a rack ready to hold the liquid you measure in the next steps. Microcentrifuge tubes are often called Eppendorf tubes. Eppendorf is a popular brand of labware.
You will be pipetting 600µL of colored sugar solution. The color helps you see how much you are measuring. Choose the correct size micropipetter and set it to 600 µL. While you are waiting to use the micropipetter practice opening and closing microcentrifuge tubes with one hand or setting the other micropipetters with one hand.
Place a tip on the end of the pipetter. Do not touch the tip with your hands. Leave it in the box and push the end of the micropipetter firmly into the tip. The smaller tips fit both the 2-20 and the 20-200 µL micropipetters. They are often yellow or clear. The larger tips are for the 200-1000 µL micropipetter and are sometimes blue.
Using one hand, hold the micropipetter and press down on the plunger with your thumb or index finger (whichever feels more comfortable). Note that there are 2 places the plunger stops. The first stop is for filling and the second stop is for delivering. Practice a few times until you can easily feel the difference between the two stops. If you are waiting to use the correct micropipetter you can practice with the other micropipettes.
Press down to the first stop. Submerge the end of the tip just under the surface of the liquid. You may rest the tip against the side of the container just under the water line to steady it. If you submerge more than just the end of the tip, liquid will collect on the sides of the tip and drip into the collection tube when you deliver it. This will result in a larger volume of liquid than was desired.
Slowly release the plunger. If you release the plunger too quickly, the liquid may splash up into the micropipetter and contaminate it. If you are pipetting viscous (thick) liquids, such as the sugar solution you are using, and you release too quickly, the liquid won’t enter the tip fast enough and your measurement will be inaccurate. Sometimes this happens with thin liquids as well, so you should always pipette slowly. Be careful not to remove the tip from the liquid before it is filled with the desired volume or you will get an air bubble in the tip and less liquid than was desired. If you released the plunger slowly and kept the tip in the liquid but you still got a bubble, you probably pushed the plunger down to the second stop instead of the first. Practice the stops again.
Without removing the tip from the beaker, dispense the liquid by pushing the plunger slowly down to the first stop. Try not to make any bubbles. Repeat step 6. Drawing up the liquid twice (in labs it is called “pipetting up and down”) can improve the accuracy of the measurement.
Dispense the liquid into 1.5 mL microcentrifuge tube and be sure it is near the 0.6 mL mark (600 µL = 0.6 mL). This is just a check to make sure you used the correct micropipetter and set it correctly. Show the instructor your tube
Discard the tip in a waste beaker by pressing the eject button. You may want to practice this technique a few times, as it is a very important skill to master.
C. Mixtures and Microcentrifuge Tube Labeling
You are going to measure different colors and amounts of water into 10 microcentrifuge tubes. Wearing gloves, choose 10 microcentrifuge tubes, close them, and label the lids 1 – 10. Always label on the top so that it can be read without removing the tube from the rack, and orient the tubes in the same direction so that you won’t confuse letters like “H” and “I” and numbers like “6” and “9”. Also, only use a permanent marker, such as a Sharpie, that will not erase or bleed if it gets wet. If your tubes are to be stored or mixed in a microcentrifuge etc., label your tubes with a group name, or your name, and the date of the experiment.
Open all of the lids of the microcentrifuge tubes so they are ready to receive the solutions. Before you begin measuring, think of what will be the most efficient way of dispensing the amount. If several of the tubes contain the same liquid, you can measure them all out before you change tips, as long as you do not touch the tip to the inside of a tube containing some other solution. Even water can be a contaminant if it changes the concentration of a given solution. You may also want to first fill all of the tubes that have the same measure of liquid so you don’t have to change the setting too often. Sometimes it matters which ingredient is added first, as is the case when diluting acids and bases.
Measure the following amounts into the indicated tubes. Mix the contents by pipetting up and down several times. DO NOT pipet so vigorously that you make bubbles. This can degrade some sensitive solutions such as enzymes, and can also contaminate the micropipetter. You may want to close the tubes as they are filled or move them back one row to avoid accidentally filling the same tube twice.
5 µL blue
100 µL clear
20 µL blue
10 µL blue
100 µL blue
500 µL clear
20 µL blue
1000 µL blue
5 µL clear
20 µL blue
1000 µL clear
20 µL blue
20 µL clear
20 µL blue
500 µL clear
500 µL blue
Check the accuracy of your measurements by setting a micropipetter to the total volume in the tube and slowly withdrawing all of the solution from several tubes. Your pipetting was accurate if you leave no solution behind and have no air bubble in your tip. The amounts in tubes 1 and 2 are so small that if any is clinging to the side you won’t be able to draw it up. If this is the case, put the tubes in a picofuge for about 10 seconds to “spin down” the liquid so it is all in the bottom of the tube. Have your instructor check your tubes before discarding them, as he or she may wish to watch you draw up the amount to check accuracy.