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5
SECTION OF BIOENGINEERING TECHNOLOGY
UniKL MICET
BIOLOGY OF CELLS
EXPERIMENT 1: INTRODUCTION TO BIOLOGY LABORATORY
OBJECTIVE:
To expose the pH meter, micropipette, spectrophotometer and weighing scale basic techniques
INTRODUCTION:
Mastery of these techniques is important for good results in experiments. Most of the
biotechnology laboratories are based on microchemical protocols that use very small volumes of
DNA and reagents. These require use of an adjustable micropipette or that measures as little as
one microliter (L) or less. The pH meter is a potentiometer that measures the potential
development between a glass electrode and a reference electrode. The glass electrode contains a
glass bulb constructed of very thin, special glass that is permeable to hydrogen ions. Adjustments
for temperature are necessary because the relationship between measured potential and pH is
temperature dependent. The spectrophotometer is utilized by molecular biologists for accurate
preparation and analysis of many types of samples. The spectrophotometer has varied
applications in the qualitative analysis of sample purity, DNA and protein quantification, cell
density measurements and assays involving enzyme-catalyzed reactions.
PART 1 (MICROPIPETTOR):
Pre-lab Preparation:
1) To simplify initial practice with a micropipettor, use coloured solutions that are easily
visible. Prepare five coloured solutions using food colouring or other dyes mixed with
water.
2) Prepare for each experiment
a) Four (4) 1.5 mL tubes, each containing 1 mL of a different colored solution, marked I,
II, III, and IV.
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10 L micropipettor + tips
Microfuge (optional)
10 mL pipet
Permanent marker
50 mL conical tube
15 mL culture tube
1.5 mL tubes
Never rotate volume adjustor beyond the upper or lower range of the pipet, as stated by
manufacturer.
Never use micropipettes without tip in place; this could ruin the precision piston that
measures the volume of fluid.
Never lay down pipettes with filled tip; fluid could run back into piston
Never let plunger snap back after withdrawing or ejecting fluid; this could damage
piston.
Pipetting Directions
1. Rotate volume adjuster to desired setting. Note change in plunger length as volume is
changed. Be sure to locate the decimal point properly when reading volume setting.
2. Firmly place proper-sized tip on end of micropipettor.
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Experiment Procedures:
(A) Small Volume Micropipette Exercise
This exercise stimulates setting up a reaction, using a micropipette with a range of 1-20 L.
1) Use permanent marker to label three 1.5 ml microcentrifuge tubes A, B, C.
2) Use matrix below as a checklist while adding solutions to each reaction tube.
3)
Tube
Sol. I
Sol. II
Sol III
Sol IV
A
4 L
5 L
1 L
B
4 L
5 L
1 L
C
4 L
4 L
1 L
1 L
Set micropipette to 4 L and add Solution I to each reaction tube.
4)
Use fresh tip to add appropriate volume of Solution II to a clean spot on reaction
tubes A, B, and C.
5)
6)
7)
Close tops. Pool and mix reagents by one of the following methods:
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 micro-centrifuge and apply a short, several second pulses. Make sure
reaction tubes are placed in a balanced configuration. Spinning tubes in a
unbalanced position will damage micro-centrifuge 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.
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Sol. I
Sol. II
Sol III
Sol IV
100 L
200 L
150 L
550 L
150 L
250 L
350 L
250 L
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pH indicator paper
Standard buffer, pH 7
Wash bottle
pH meter
Procedures:
Part A: Visual Estimation of pH
1. Prepare 0.1 M solutions (100 mL) of K2HPO4 and KH2PO4.
2. Set up a series of twelve 18 150 mm test tubes as shown in Table A-1.2A.
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7 of 10
Questions
1. Show your calculations for preparing the following solutions:
a. 200 mL of 20% (w/v) NaOH
b. 1 L of 1.0 M Tris (MW 121.1 g/ mol)
c. 100 mL of 0.2 M EDTA (MW 372.2 g/mol)
2. How much of the above Tris and EDTA solutions were 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.
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Procedure:
1.
Warm up (for about 20 min) the spectrophotometer set at 540 nm before use.
2.
3.
Use micropipette to add to each successive tube the following amounts of bromophenol
blue (1.25% w/v): 0.5, 1, 2, 4, 10, 20, 50 and 100 L.
4.
5.
6.
Transfer the above dye solutions from least concentrated to most concentrate into the
same cuvette.
7.
Record the readings and graph the results [Absorbance at 540 nm (Ab540) vs concentration
of bromophenol blue, (w/v)].
Questions:
1. Consistency of micropipette usage depends on strict attention to what operational
procedures?
2. Explain the relationship among absorbance value, optical density, and percent
transmittance.
Weighing balance
Do not try to calibrate the instrument without prior permission of student in charge.
Do not subject the table carrying weighing balance to severe vibrations or shocks, which
may affect the calibration.
If you spill something on the weighing pan, do not rush over to clean it. Contact the
student in charge of the instrument regarding cleaning.
Procedure:
1. Clean balance before use.
2. Turn on the balance scale.
3. Place the container on balance.
4. Tare the balance.
5. Place the sample in container and note down the weight.
6. Clean balance after use.
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