Lab 1 Essential Lab Skills-MB Ariza PDF

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Lab 1 Essential Lab Skills-MB Ariza...

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Lab 1: Essential Laboratory Skills: Pipetting Skills, & Proper Use of Glassware Purpose: You will write your own purpose and goals. Hint: This laboratory activity introduces the proper use of the micropipette; a tool used throughout molecular biology and biotechnology protocols (procedures) that follow these general technique labs. Learning the proper use of this tool will be important for obtaining good results.

Introduction: There are few rules to maintain an efficient pipetting technique in a laboratory. It is highly recommended to watch the following YouTube videos as to how to use micropipettor: http://www.youtube.com/watch?v=kCm2t1SO2IU http://www.youtube.com/watch?v=tL0acTneiNY A. Know your pipet If it is a pipetman micropipette (Corning), know its volume range and how to adjust that range. Most pipetman have two stop-points on the plunger. Fit the tip to the end of the shaft and press down to ensure an airtight seal. When filling the pipet, press the plunger to the first stoppoint, insert the pipet tip in the solution (Not too deeply into the solution!) and slowly release the plunger. If you release the plunger too quickly, the liquid may splash up into the micropipet and contaminate it. If you are pipetting viscous (thick) liquids, such as dense glycerol solution, 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. Do not insert the pipet tip deep into the solution to draw the solution. When emptying the pipette tip, put the tip into the bottom of container, press down on the plunger and go beyond the first stop-point to the second final stop-point to expel the fluid. B. General diagram of a typical micropipettor Micropipettors come in various sizes ranging from 0.2 µl capacity to 5000 µl capacity. Generally, 20 µl, 200 µl, and 1000 µl size are most commonly used in molecular biology laboratory activities. The volume indicator is read from top to bottom. For 20 µl capacity and 200 µl capacity micropipettors, black digits indicate microliters and red digits indicate tenths and hundredths of microliters. For 1000 µl capacity micropipettors, red digits indicate milliliters and black digits indicate microliters. Not all micropipettors have colored digits on their volume indicators. (Diagram by B. Ball)  BIOT 5021 MB LAB ARIZA

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The American Biology Teacher. 66(4): 291–296. 2004. Page 1

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C. Make sure that the pipet is securely placed on the pipetman If the tip is loose on the pipet, the volume will not be correct and the tip may fall down. If you are working with a power-assisted pipet-aid to deal with larger volumes (> 5 ml), the upper button fills the pipet and the lower button expels the fluid. Do not draw the fluid beyond the upper marking of the pipet because this will wet the cotton in the neck of the pipet and the pipet will no longer work.

D. When working under sterile condition, the following rules apply •

Anything that your hand (even gloved hand) touches is no longer sterile. If your hand passes over an open sterile plate or open sterile plate or bottle, it is likely that you have contaminated these items and the solution they contain.

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If the pipet tip touches anything other than a sterile surface, it is no longer sterile.

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Keep your workspace in good order. A messy workspace could cause contamination of your experiment.

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When transferring sterile fluids from a stock bottle, remove the cap with one hand and insert the pipet at a slight angle into the bottle without touching the outside of the bottle with the pipet tip. While transferring the fluid to the plate or tube, replace the lid on the bottle. If multiple transfers are needed from one bottle to several plates or tubes, the lid is placed on the bottle loosely and replaced after all transfers. Do not pass your hand over the open bottle

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If someone is working with sterile materials, do not interfere with work. Ask permission and give them time to finish what they are doing and to close the containers.

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Keep this in mind when working in a laboratory, work as a team!

E. Organizing Your Work Space When your work requires aseptic (sterile) conditions, you should wash the bench top with 70% ethanol. Although this procedure does not need to be performed in sterile conditions, clean 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

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items you are not immediately using. Always have a waste beaker for used tips when you are micropipetting, do not use the sink for the disposal of tips!

F. Calibrating Lab Equipment Different pieces of lab equipment are designed to measure properties such as temperature, pH, mass, and volume to varying degrees of accuracy. If the temperature markings on the side of a thermometer are not set accurately, the instrument’s measurements will not be accurate. The accuracy of these markings is due to the calibration or standardization of the thermometer. The standards used to calibrate a thermometer are freezing and boiling water. Some equipment must be periodically recalibrated because the settings are not as immovable as lines on a graduated cylinder or thermometer. The calibration of instruments such as pH meters, electronic balances, and micropipettes can be rendered inaccurate by factors such as movement, humidity, electrical field changes, and many others. These instruments should be calibrated each time they are turned on. If they are left on for long periods of time or used frequently during a day, they should be periodically recalibrated during that time as well.

Materials and Equipment: •

25-mL and 100 mL beakers

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10 mL and 50 mL graduated cylinders

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25 mL flasks

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5 mL serological pipettes

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10 mL serological pipettes

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Pipet aid

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P20, P100, P1000 and tips

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2.0 mL microcentrifuge tubes

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PCR strips

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Colored solutions

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Weighting boats and Parafilm

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Sharpie

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Distilled water

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Tube racks

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Electronic balance and calibration weight

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Vortex mixer

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Procedures: A. Calibrating and Using an Electronic Balance The standards used to calibrate electronic balances are objects of known mass. For balances that measure to ± 0.01g, the standard is usually a 200-gram weight. These balances are used to measure amounts over 0.05 g. When you place the 200-gram weight on the balance in calibration mode, the balance recognizes the weight as 200 grams, and will then use that information to measure other masses. 1. Dispense 50 mL of yellow stock solution into a 100- or 150-mL beaker.

2. Use the 200-gram weight to verify the balance accuracy and record the weight. 3. Place a weight boat on the balance pan and press the tare button. This subtracts the weight of the weigh boat so that you are only weighing what is put inside it. 4. Draw up 4 mL of yellow solution using a 5 mL serological pipette and deliver it into the weigh boat. You might need to practice drawing up and delivering the liquid back into the flask until you can do it smoothly. Remember to discard the remaining liquid in your waste beaker, not back into the stock solution. 5. Record the mass of the yellow solution in your notebook and in the results section below (step 7). At sea level, 1.00 mL of water weighs 1.00 g. This should help you decide if your measurement was accurate. 6. Have each member of the group repeat step 4 and add 4 mL of blue water to the weigh boat (do not empty the weigh boat between measurements). Divide the total mass by the number of people in the group to find the average mass. Show calculations in your notebook. 7. Answer the following questions: a. What does it mean to “tare” a balance? b. What is a blowout, or serological pipet? How does this compare with a Mohr, or todeliver, pipet? c. The definition of a gram is the mass of 1 mL of pure water at 20 " C (about room temperature) and 1 atmosphere of pressure.

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i.

What should be the average mass of the water measured? Show your calculations. ______ g

ii. What was the average mass of the water your group measured? Show your calculations. _______ g iii. If there is a difference between the predicted mass (A) and the observed mass (B), how can you account for it?

B. Measuring Accuracy of Glassware 1. Measure 10 mL of water in a 50 mL beaker using the lines on the beaker for your measurement. Tare a weigh boat on the balance. Pour the water in the weigh boat and record the mass in your lab notebook and on step 7. Each member of the group should repeat this step and record each measurement. Calculate the average mass of 10 mL of water measured with a beaker.

# 2. Repeat step 1 using a 10 mL graduated cylinder 3. Repeat step 1 again with a 50 mL or 100 mL graduated Cylinder. 4. Repeat step 1 using a 10 mL pipette. 5. Repeat step 1 using a 25 mL flask. 6. Clean up the glassware. Pour the contents of the waste beaker down the sink with plenty of water, and return dirty glassware to the sink. Wipe off your lab bench with a wet paper towel. Make sure that the balances, and the areas around them are clean and dry, and turn off the equipment. 7. Record the average mass of the water you measured (include units): a. 50 mL Beaker _______________ b. 10 mL graduated cylinder __________ c. 50 or 100 mL graduated cylinder _____________ d. 10 mL pipette ______________ e. 25 mL flask _______________ f.

Which of these measures was most accurately?

g. Will it probably always be more accurate? ______ Why or why not?  BIOT 5021 MB LAB ARIZA

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C. Pipetting Practice Pipetting error is a major contributor to poor laboratory results. Two important sources of pipetting error are the use of an uncalibrated micropipet and the practice of inaccurate pipetting. Practice setting the volume on the micropipette; 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 plunger button top of the micropipet to identify its measuring range. Remember that the value listed on the top is the largest volume you can measure on that pipet and that the lowest volume is 1/10 of the largest volume. On a P1000 micropipet, the largest and lowest measurable volume are 1000 and 100 µL, respectively; 200 and 20 µL on a P200 micropipet; 20 and 2 µL on a P20 micropipet. 1. Set a P1000 pipet to 0.45 mL (450 µL), a P200 micropipettor to 0.15 mL (150 µL), and a P20 micropipettor to 0.015 mL (15 µL). You should practice doing that kind of conversion in your head. 2. Insert a tip into the end of the pipet by pushing the end of the micropipet firmly into the tip in the box. Do not touch the tip with your hands. The smaller tips fit both the P20 and the P200 micropipette. They are often yellow or clear. The larger tips are for the P1000 micropipet and are often blue or clear. 3. Take up the water volume (microliter) you want, empty into a waste beaker, and 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.

D. Checking Pipette Calibration 1 mL H2O = 1.0 (0.995 – 0.998) g at 25°C 1. Check the pipet calibration using an electronic analytical balance by: a. Place a strip of parafilm on the balance pan b. Set the balance to zero (tare) c. Dispense the water onto the balance and read the readings for the volumes below

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Table 1: Pipet Calibration Pipet

Volume (µl)

Volume (ml)

Expected weight (g)

P20

10.0

0.010

P200

100.0

0.100

P1000

500.0

0.500

Recorded weight (g)

E. Practice Accuracy and Precision Accuracy is how close a measurement value is to the true value or accepted value, referring to a mean value of measurements. Precision is the consistency of a series of measurements or tests, referring to standard deviation. Familiarize yourself with the amount of liquid in a tip. Try to develop a sense for the volume of liquid you are pipetting by looking at the amount of liquid in the tip. It is easy to grab the wrong pipet or to set it incorrectly; however, you should be able to tell the difference between 1 µL in a tip and 10 µL in a tip just by looking at the amount of liquid in the tip. Each member of the group should complete the following exercises individually. I.

Small-volume micro-pipetting exercise 1. Obtain a P20 micropipet, appropriate tips and 2 microfuge tubes 2. Label the microfuge tubes A and B and your name initials. 3. Tare the balance and record the weight of each tube Table 2: Small Volume Pipetting Tube

Red (µl)

Blue (µl)

Green (µl)

Water (µl)

A

---

2.0

2.0

11.0

B

3.0

2.0

---

10.0

Total Expected Vol. (µl)

Total Measured Vol. (µl)

Color Observed

4. Use the table above as a guide for adding appropriate volumes of the colored solutions to each tube. 5. First add the appropriate amount of water to the bottom of each tube. Be sure all of the liquid comes out and forms a small bubble of liquid at the bottom of the tube. 6. Next add the colored solutions one at a time. Dispense the solution directly into the small bubble of liquid at the bottom of the tube.  BIOT 5021 MB LAB ARIZA

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7. After you have added all of your solutions into each tube, practice mixing the contents with a micropipet. Set your micropipet to 15 µl and slowly pipet the mixture up and down until well mixed. 8. Place all the tubes in the vortex mixer and apply a short (1-2 seconds) pulse. 9. Record the final color of the solution in each tube in the table above. 10. Close the tubes and record their weight. 11. Calculate the accuracy of your pipetting as follows:! Accuracy (% error) = 100 x (Measured Volume - Expected Volume)/Expected Volume 12. Record results in the lab notebook. II. Large-volume micro-pipetting exercise 1. Obtain a P200 and P1000 micropipettes, corresponding tips, and 4 microcentrifuge tubes 2. Label the tubes C, D, E, F. 3. Tare the balance and record the weight of each tube 4. Use the table below as a guide for adding appropriate volumes of the colored solutions to each tube. Table 3: Large Volume Pipetting Tube

Red (µl)

Blue (µl)

Green (µl)

Water (µl)

C

40

---

40

920

D

---

20

260

720

E

---

120

20

860

F

60

30

30

880

Total Expected Vol. (µl)

Total Measured Vol. (µl)

Color Observed

4. Mix well (by finger vortexing or micropipetting) and place all the tubes in the vortex mixer and apply a short (1-2 seconds) pulse. 5. Record the final color of the solution in each tube in the table above. 6. Close the tubes and record their weight. 7. Calculate the accuracy of your pipetting as follows:! Accuracy (% error) = 100 x (Measured Volume - Expected Volume)/Expected Volume 8. Record results in the lab notebook.  BIOT 5021 MB LAB ARIZA

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III. Precision micro-pipetting exercise 1. Obtain a strip of PCR tubes and rack. Label the tubes 1-8. 2. Tare the balance and record the weight of the empty strip and rack. 3. Using the P200 pipette 150 µl into the 1st tube. 4. Record the weight of the PCR strip. 5. Repeat steps 3 and 4 for the rest of the tubes. Make sure to record the weight of the PCR strip after each pipetting. 6. Complete the percent error, average error and standard deviation in data table 4. 7. Use the formulas below to evaluate the precision (standard deviation) of your pipetting. 8. Record results in the lab notebook. Table 4: Precision Practice Sample

1

2

3

4

5

6

7

8

Actual Weight (g) Expected Weight (g) % Error Average Error

% Error = [(actual value – expected value) ÷ expected value] x 100 ! Average error = (∑ error) ÷ (# trials)! 2 1/2 Standard deviation = ((x-u) / (N-1) ) ! ∑ = sum# x = % error# u = average error# N = total number of errors calculated (in our case 8) ! 1⁄2 = Square root of the total sum

# 9. Analyze your data. Does the standard deviation indicate a significant variation in the data? If yes, do you believe that this variation is due to the accuracy of the micropipette or due to poor technique? Explain your answer by using the data to support your conclusion.

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