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BIOCHEMISTRY
Experiment 1
An Introduction to Biology Laboratory
Objectives
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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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
Experiment Procedures
Questions
1. List 3 precautious to take while using micropipettor.
2. Discuss care and maintenance of micropipettor.
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.
REAGENTS / SUPPLIES
Procedure
Questions:
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