Biochemistry Laboratory Manual Arti S Pandey, Arun Pandey, Naveen K Shreevastava, Durga P Neupane
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MBBS I
  • Use of Micropipettes
  • Buffers and pH
  • Qualitative tests for Carbohydrates
  • Qualitative Tests for Proteins
  • Separation of Serum Proteins by Gel Electrophoresis
  • Principles of Colorimetry
  • Estimation of Serum Total Protein and Determination of Albumin to Globulin Ratio
  • Genomic DNA Isolation, Amplification of the D1S80 Locus and DNA-Gel Electrophoresis
  • Estimation of Blood Glucose by GOD-POD Method
  • Estimation of Serum Total Cholesterol by Modified Zak's Method
  • Estimation of Serum Triglycerides
  • Estimation of Serum Uric Acid by Caraway's Method2

Use of MicropipettesExperiment 1

 
INTRODUCTION
Pipettes are used for measuring liquid volume with high precision. They are primarily used for transferring fluid. The pipette is filled by suction provided by the mouth, a rubber ball or a mechanical device at one end designed for this purpose. Mouth pipetting must not be carried out in a clinical laboratory.
The advancement in biochemical techniques allow for tests to be carried out on microliters of human samples. Such small volumes depend on precision of measuring instruments which give minimum error. Micropipettes are the standard laboratory instruments used to measure and transfer small volumes of liquid. They have the following parts as shown in Figure 1.1:
  1. Plunger button
  2. Tip ejector button
  3. Volume adjustment dial
  4. Digital volume indicator
  5. Shaft
  6. Attachment point for a disposable tip.
Liquids are not drawn directly into shaft of the pipette. Instead, disposable tips are attached to the shaft.
 
USE
Micropipettes are generally used to measure volumes of 1 ml or less. Because they measure such small volumes, they are calibrated in microliters (µl) rather than milliliters. Different micropipettes 4are available to measure different volumes, and should be chosen in such a way that the volume being measured falls within the available range, e.g. for measuring 50 µl of a liquid, use the pipette with the range 10 to 100 µl instead of using the one with range 2 to 20 µl three times with 20 µl, 20 µl and 10 µl to add upto 50 µl.
zoom view
Figure 1.1: Micropipette and disposable micropipette tips (1 ml and 100 µl)
 
PROCEDURE FOR MICROPIPETTING
  1. Choose the appropriate micropipette.
  2. Adjust to the correct volume.
  3. Insert tip on the shaft.
  4. Depress the thumb knob to the first stop.
  5. Immerse the tip approximately 3 mm into the sample solution.
  6. Slowly release the thumb knob to the initial position. Watch as the solution is drawn up slowly into the tip. Do not release the plunger too quickly.
  7. 5Withdraw the tip from the sample solution.
  8. Place the tip against the side wall of receiving container.
  9. Smoothly depress the thumb knob to the first stop. Then depress the knob to the second stop.
  10. Remove the tip from the receiving container and return knob to the initial position.
  11. Remove the disposable tip by firmly depressing the tip ejector knob.
  12. Add a new tip and continue.
  13. If you need to pipette a different solution, change the tip.
Note: If air gets into the tip during aspiration, do steps “g” to “i” and start from step “e” again.
 
CHECKING YOUR PIPETTING SKILL
Good pipetting is indicated by uniformity of the volume of liquid dispensed.
  1. Measure the appropriate volume of liquid using micropipette.
  2. Put a waterproof paper on an electronic balance and adjust the reading to zero (0). Dispense the liquid and note the reading.
  3. Repeat the same procedure 3 to 5 times.
  4. Take the average weight of liquid measured and find the standard error using a calculator or the formula provided.
 
Observations
Volume of liquid measured …………μl
Weight of liquid measured
  1. (x1) =
  2. (x2) =
  3. (x3) =
  4. (x4) =
  5. (x5) =
Mean weight (m) = ________________
Standard deviation (SD) =
6
where N=total number of observations
Standard error (SE) = SD/√N = ________________
Volume measured= Mean ± SE = ____________ µ l