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BIOCHEMISTRY

Experiment 1
An Introduction to Biology Laboratory

Objectives

 To expose the pH meter, micropipette, spectrophotometer and weighing scale basic


techniques used throughout this course. Mastery of these techniques is important for
good results in all of the 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 micropipettor that measures as little as
one microliter (µl). The pH meter is a potentiometer that measured 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. This exercise is designed to familiarize students with this instrument.
Spectrophotometer has varied applications in the qualitative analysis of sample purity,
DNA and protein quantitation, cell density measurements and assays involving
enzyme-catalyzed reactions.

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
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.
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Supplies and Equipment


 10 µl micropipettor + tips
 100 µl micropipettors + tips
 1000 µl micropipettors + tips
 10 ml pipet
 Pipet aid or bulb
 50 ml conical tube
 15 ml culture tube
 1.5 ml tubes
 Beaker for waste/used tips
 Bunsen burner (optional)
 Microfuge (optional)
 Permanent marker
 Test tube rack

Use of Digital Micropipettors

NEVERS
 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.
 Never immerse barrel of pipettor in fluid.
 Never flame micropipettor tip.
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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.
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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.
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 tip 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. Small Volume Micropipettor Exercise


This exercise stimulates setting up a reaction, using a micropipettor with a range
of 1.5 – 10 µl or 1 – 20 µl.
1) Use permanent marker to label three 1.5 ml tubes A, B, C.
2) Use matrix below as a checklist while adding solutions to each reaction tube.

Tube Sol. I Sol. II Sol III Sol IV


A 4 µl 5 µl 1 µl -
B 4 µl 5 µl - 1 µl
C 4 µl 4 µl 1 µl 1 µl
3) Set micropipettor to 4 µl and add Solution I to each reaction tube.
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4) Use fresh tip to add appropriate volume of Solution II to a clean spot on


reaction tubes A, B, and C.
5) Use fresh tip to add 1 µl of Solution III to tubes A and C.
6) Use fresh tip to add 1 µl of Solution IV to tubes B and C.
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 in
the micro-centrifuge rotor. Spinning tubes in an unbalanced
position will damage microfuge motor.
8) A total of 10 µl of reagents was added to each reaction tube. To check that
your measurements were accurate, set pipette to 10 µl 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.
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B. Large Volume Micropipettor Exercise


It is easier to measure when using a large-volume micropipettor. However if
the plunger is not released slowly, an air bubble may form or solution may be
drawn into piston.
1. Use a permanent marker to label two 1.5 ml reaction tubes E and F.
2. Use matrix below as a checklist while adding solutions to each reaction tube.

Tube Sol. I Sol. II Sol III Sol IV


E 100 µl 200 µl 150 µl 550 µl
F 150 µl 250 µl 350 µl 250 µl

3. Set micropipettor to add appropriate volumes of Solutions I-IV to


tubes E and F. Follow same procedure as for small-volume pipettor.
4. A total of 1000 µ1 of reactants was added to each tube. To check that your
measurements were accurate, set micropipettor to 1000 µ1 and carefully
withdraw solution from each tube.
a. Is the tip just filled?
or
b. Is a small volume of fiuid 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.)
5. If measurements were inaccurate, repeat exercise to obtain a near-perfect
result.

Questions
1. List 3 precautious to take while using micropipettor.
2. Discuss care and maintenance of micropipettor.

PART II (pH METER)


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Reagents/ Supplies
 Beakers (250 ml)
 Bromothymol blue (0.25% w/v)
 pH indicator paper
 pipets (1, 5 and 10 ml)
 Potassium Phosphate, dibasic ( Dipotassium Phosphate, K2HPO4)
 Potassium Phosphate, monobasic (Monopotassium Phosphate, KH2PO4)
 Standard buffer, pH 4.01
 Standard buffer, pH 7
 Test tubes 18 × 150 mm
 Volumetric flasks 100ml
 Wash bottle

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.
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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.
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.
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4. Record your observations from Part A and B. Correlate the measured values
from Part B to the expected pH value from Table A-1.2 A.

Questions
1. Explain the basic principles of pH meter.
2. 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).
3. 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)?
4. Describe the relationship between buffer working range and its pK value.
5. Discuss the term buffer capacity.

PART III: (SPECTROPHOTOMETERS)

Figure 1: diagram of components of spectrophotometers

REAGENTS / SUPPLIES

 Bromophenol blue (1.25% w/v)


 Cuvettes (alternatively colorimeter tubes if needed)
 Micropipettors and tips
 Pipets, 10 ml
 Test tubes, 18 × 150 mm
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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
bromophenol blue (1.25% w/v): 0.5, 1, 2, 4, 10, 20, 50 and 100 µl.
manufacturer instructions for use of micropipettors should be followed
scrupulously.
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. Explain the basic principles of spectrophotometer.


2. What is the use of spectrophotometer in the field of biochemistry?
3. Explain the relationship among absorbance value, optical density, and percent
transmittance.
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PART IV (WEIGHING BALANCE)

REAGENTS/ SUPPLIES
 Weighing balance
 Beakers (250 ml)

DON’TS
 Don’t move the weighing balance in any case.
 Don’t try to calibrate the instrument without prior permission of student in
charge.
 Don’t change the configuration of the instrument.
 Don’t subject the table carrying weighing balance to severe vibrations or shocks,
because it can affect the calibration.
 If you spill something on the weighing pan, don’t rush over to clean it. Contact
the student in charge of the instrument regarding cleaning.

Procedure

1. Basic weighing function:


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.