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PART 1 ( MICROPIPETTOR)
Pre-lab Preparation:
1) To simplify initial practice with a micropipettor, use colored solutions that are easy
visible. Prepare five (5) colored solutions using food coloring or other dyes mixed with
water.
2) Prepare for each experiment
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a) Four (4) 1.5 ml tubes, each containing 1 ml of a different colored solution, marked I,
II, III, and IV.
b) One 50 ml conical tube containing 25 ml of the colored solution marked V.
Supplies and Equipment
10 l micropipettor + tips
10 ml pipet
50 ml conical tube
15 ml culture tube
1.5 ml tubes
Microfuge (optional)
Permanent marker
Never rotate volume adjustor beyond the upper or lower range of the pipet, as stated by
manufacturer.
Never use micropipettor without tip in place; this could ruin the precision piston that
measures the volume of fluid.
Never lay down pipettors with filled tip; fluid could run back into piston
Never let plunger snap back after withdrawing or ejecting fluid; this could damage
piston.
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Pipetting Directions
1. Rotate volume adjustor to desired setting. Note change in plunger length as volume is
changed. Be sure to located decimel point properly when reading volume setting.
2. Firmly seat proper-sized tip on end of micropipettor.
3. When withdrawing or expelling fluid, always hold tube firmly between thumb and forefinger.
Hold tube at nearly eye level to observe the change in the fluid level in pipet tip. Do not pipet
with tube in test tube rack or have another person hold tube while pipetting.
4. Each tube must be held in the hand during each manipulation. Grasping the tube body, rather
than the lid, provides more control and avoids contamination from the hand.
5. Hold pipettor almost vertical when filling
6. Most digital micropipettor have a two-position plunger with friction stops.Depressing to
the first stop measures the desired volume of air to blow out any solution remaining in the
tip. Pay attention to these friction stops, which can be felt with the thumb.
7. To withdraw sample from reagent tube:
a. Depress plunger to first stop and hold in this position. Dip tip into solution to be
pipetted, and draw fluid into tip by gradually releasing plunger.
b. Slide pipet tip out along inside wall of reagent tube to dislodge excess droplets
adhering to the outside of tip.
c. Check that there is no air space at the very end of the tip.To avoid future pipetting
errors, learn to recognize the approximate level that particular volumes fill the pipet
tip.
8. To expel sample into reaction tube:
a. Touch pipet tip to inside wall of reaction tube into which the sample will be emptied.
This creates a capillary effect that helps draw fluid out of tip.
b. Slowly depress plunger to the first stop to expel sample. Depress to second stop to
blow out last bit of fluid. Hold plunger in depress position.
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c. Slide pipet out of reaction tube with plunger depressed to avoid sucking any liquid
back into tip.
d. Manually remove or eject tip into a beaker kept on the lab bench for this purpose. The
tip is ejected by depressing a separate t ip ejection button.
9. To prevent cross contamination of reagents.
a. Always add appropriate amounts of single reagent sequentially to all tubes.
b. Release each reagent drop onto new location on inside wall, near bottom of reaction
tube. In this way, the same tip can be used to pipet the reagent into each reaction tube.
c. Use fresh tip for each new reagent to be pipette.
d. If tip becomes contaminated, switch to a new one.
10. Eject used tips into a beaker kept on the lab bench for this purpose.
Experiment Procedures
A.
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a. Sharply tap tube bottom on bench top. Make sure that the drops have
pooled into one drop at the bottom of the tube.
Or
b. Place in microfuge and apply a short, several second pulses. Make sure
reaction tubes are placed in a balnced configuration in the microfuge rotor.
Spinning tubes in a unbalanced position will damage microfuge motor.
8) A total of 10 l of reagents was added to each reaction tube. To check that your
measurements were accurate, set pipette to 10 l and very carefully withdraw
solution from each tube.
a. Is the tip just filled?
Or
b. Is a small volume of fluid left in tube?
Or
c. After extracting all fluid, is an air space left in tip end? (The air can be
displaced and actual volume determined simply by rotating volume
adjustment to push fluid to very end of tip. Then, read volume directly.)
9) If several measurements were inaccurate, repeat exercise to obtain a near-perfect
result.
B.
Sol. I
100 l
Sol. II
200 l
Sol III
150 l
Sol IV
550 l
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F
3.
150 l
250 l
350 l
250 l
pH indicator paper
Standard buffer, pH 7
Wash bottle
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Equipment
pH meter
PROCEDURE
PART A: Visual Estimation of pH
1. Prepare 0.1 M solutions (100ml) of K2HPO4 and KH2PO4.
2. Set up a series of twelve 18 150 mm test tubes as shown in Table A-1.2A.
3. To tubes 1, 3, 5, 7, 9, and 11, add 5 drops of bromthymol blue and mix. You now have a
series of color standards covering the pH range of 5.3 to 7.73. Record your observations.
This exercise simply illustrates that indicator dyes may be useful as pH indicators.
Section A
1. Standardize the pH meter using the standard pH 7 buffer. Rinse the electrodes using a
wash bottle. Do not wipe the electrodes with tissues because this creates a static electric
charge on the electrodes and may cause erroneous readings. Remove the last drop of
water by carefully touching a piece of clean tissue paper to drop.
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2. Measure the pH of the standard pH 4.01 buffer. Reset the pH meter if necessary. It is
most important to measure the pH with two standard buffers to ensure that the pH meter
is functioning properly over the entire pH range.
3. Measure the pH of the six solutions in tubes 2, 4, 6, 8, 10 and 12 (prepared in Part A)
with the pH meter. Rinse the electrodes between readings and handle them carefully. You
may also wish to use pH indicator paper to get an idea of the pH of the solutions. this
rapid method is often accurate enough for some applications and is especially useful for
very small volumes or radioactive solutions.
4. Record your observations from Parts A and B. correlate the measured values from Part B
to the expected pH value from Table A-1.2 A.
Questions
1. Show your calculations for preparing the following solutions:
200 ml of 20% (w/v) NaOH, 1 liter of 1.0 M Tris (MW 121.1 g/ mol) and 100 ml of
0.2 M EDTA (MW 372.2 g/mol).
2. How much of the above Tris and EDTA solutions is used to prepare 100 ml of TE
buffer (10 mM Tris and 1 Mm EDTA)?
3. Describe the relationship between buffer working range and its pK value.
4. Discuss the term buffer capacity.
PART III: (SPECTROPHOTOMETERS)
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REAGENTS / SUPPLIES
Procedure
1. Warm up (about 20 minutes) the spectrophotometer set at 540 nm or a Klett colorimeter
(green filter, wavelength range of 500 to 570 nm) before use. The Klett reading can be
converted to optical density (OD) by multiplying the Klett reading by 0.002.
2. Place 10 ml of distilled water in each of eight test tubes.
3. Use micropipettors to add to each successive tube the following amounts of bromphenol
blue (1.25% w/v) : 0.5, 1, 2, 4, 10, 20, 50 and 100 l. manufacturer instructions for use of
micropipettors should be followed scrupulously (see Appendix 5, use of micropipettors).
4. Vortex each tube until the dye is in solution.
5. Set the spectrophotometer/ colorimeter to zero with distilled water.
6. Transfer the above dye solutions from least concentrated to most concentrate into the
same cuvette or Klett tube from reading to reading.
7. Record the readings and graph the results.
Questions:
1. What may account for difference in OD values obtained using Klett colorimeter with a
green filter as compared to a spectrophotometer set at 540 nm?
2. Consistency of micropipettor usage depends on strict attention to what operational
procedures?
3. Explain the relationship among absorbance value, optical density, and percent
transmittance.
PART IV (WEIGHING BALANCE)
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REAGENTS/ SUPPLIES
Weighing balance
Dont subject the table carrying weighing balance to severe vibrations or shocks, because
charge.
If you spill something on the weighing pan, dont rush over to clean it. Contact the
student in charge of the instrument regarding cleaning.
Procedure