# Use of Micropipettes

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1 Use of Micropipettes Prior to lab you should understand: The function of micropipettes in the laboratory Basic parts of micropipette What volumes are measured with P, P and P1 micopipettors How to read the volume indicator on a P, P and P1 How much each micropipettor costs and how they are paid for I. Objective: Learn how to use a micropipettor II. Background: Micropipettors are the standard laboratory equipment used to measure and transfer small volumes of liquids. You will use them throughout this semester and in advanced courses that you take in the future. It is essential that you master their use if you are to be successful in your experiments. A. Parts of a micropipette a. Plunger button b. Tip ejector button c. Volume adjustment dial d. Digital volume indicator e. Shaft f. Attachment point for a disposable tip c d e a b B. Three sizes of micropipettes f The micropipettors in this laboratory come in three different sizes each of which measures a different range of volumes. The three sizes are P, P and P1. These sizes are noted on the top of the plunger button. Size Micropipette P P P1 Range of volumes measured.5-µl -µl 1-1µl

3 Practice In the boxes below, write how many µls the following digital readout correspond to in each of the pipetters? 7 P Digital Readout P P1 Write volume in µl Which micropipettor would be appropriate to measure 5µl? Fill in the numbers that should appear in the digital display if that pipettor were to measure 5µl. D. Pipette Tips Liquids are never drawn directly into the shaft of the pipette. Instead, disposable plastic tips are are attached to the shaft. There are two sizes of tips. The larger blue tips are used for the P1. The smaller clear tips are used for the P and P. The tips are racked in plastic boxes with covers. When you receive a box, it will be sterile. Please be careful when touching box or tips not to contaminate them. The box should be closed when not in use to prevent airborn contamination. Inserting the Tip 1. Select the correct size tips.. Open the box without touching the tips with your hands.. Insert the micropipette shaft into the tip and press down firmly. This will attach the tip to the shaft. 4. Remove the micropipettor with the tip attached. 5. Close the box without touching the tips with your hands. E. Punger Settings The plunger will stop at two different positions when it is depressed. The first of these stopping points is the point of initial resistance and is the level of depression that will result in the desired volume of solution being transferred. The second stopping point is when the plunger is depressed beyond the initial resistance until it is in contact with the body of the pipettor. At this point, the plunger cannot be depressed further. This second stopping point is only used for the complete discharging of solutions from the plastic tip.

4 F. Measuring and transferring a volume of liquid Before measuring and transferring liquid: Choose the appropriate size micropipettor Adjust to the correct volume Insert tip on the shaft. Measuring and transferring liquid The figure below shows the correct operation of the pipetteman. Important: note the first plunger stop is used in steps 1 through 4. The second plunger stop is only used in step 5. Depress the thumb knob to the first stop. Immerse the tip approximately mm into the sample solution (step 1). Slowly release the thumb knob to the initial position (step ). Watch as the solution is drawn up slowly into the tip. Do not release the plunger too quickly. Rapid release might draw bubbles in the solution and might splash solution on the non-sterile shaft. Withdraw the tip from the sample solution. Place the tip against the side wall of the receiving container (step ). Smoothly depress the thumb knob to the first stop (step 4), pause, then depress the knob to the second stop (step 5). Remove the tip from the receiving container, and return knob to the initial position. Do not let the knob snap back. Remove the disposable tip by firmly depressing the tip ejector knob (step 6). Add as new tip and continue.

6 IV. Procedure: A. Preparing a dilution of Blue dye 1. Choose appropriate micropipettor to measure 75µl.. Transfer 75 µl of blue dye to empty microfuge tube.. Use a P1 to transfer 1ml of water to microfuge tube. 4. Mix tube vigorously B. Spectrophotometer reading 1. Pour the blue dye you diluted in the microfuge tube into a cuvette. Insert cuvette into spectrophotometer. Observe absorbance reading from digital display on spectrophotometer 4. Record measurement in data sheet below C. Comparison to instructor value 1. Obtain correct absorbance value for 75 µl dilution from instructor. Divide your value by the instructor s value to determine whether you were within 1% of the instructor s value. If you are outside the 1% range you are making an error in your pipetting. D. Independent trials 1. Repeat 75µl dilution two more times as outline in boxes A-C.. Rinse and tap dry cuvette between each measurement.. Use a new microfuge tube for each dilution 4. Record values in data collection sheet. 5. Compare with instructors value for same dilution Repeating with other dilutions 1. Repeat procedures as outlined in boxes A-D except as outlined below.. Prepare new dilutions using the following quantities of blue dye a. µl b. 15µl c. 5µl. Record your data on the table collection sheet attached. 4. Conduct three trials for each dilution

7 V. Short Report Name: Due at end of class session Data Collection Sheet Dilutions Trials Student Absorbance Instructor Absorbance Ratio (S/I) 75µl 1 µl 1 15µl 1 5µl 1

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