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CELL AND MOLECULAR

BIOLOGY LABORATORY
PRELIMINARY PERIOD
EXPERIMENT 1
Use of Micropipettor and
Spectrophotometer
Micropipettor

1. The volume of air space in


the barrel is adjusted by
screwing the plunger further in
or out of the piston
2. The volume is displayed on
the digital readout
3. Depressing the plunger will
displace the specified volume
of air from the piston
4. Releasing the plunger creates
a vaccum, which will draw
an equal volume of fluid
into the tip
5. Withdrawn fluid is expelled by
depressing the plunger
- If there are air bubbles,
depress the plunger and
withdraw needed amount of
volume (again)
Types of Micropipettors
1. Red
- range is 0.5-1.0 uL
- tip: 10 uL
2. Yellow
- range is 10-100 uL
- tip: 100 uL
3. Blue
- range is 1000-1000 uL
- tip: 1000 uL

Micropipettor is used to
transfer volumes that are less
than 1mL
Measures as little as one
microliter (uL), one millionth
(10^-6) of a litter
Most useful units of liquid
measurement in molecular
biology are milliliter (mL) and
microliter (uL)
Micropipettor is a precision
pump with a disposable tip

How to use the Micropipettor

USING THE MICROPIPETTOR:

Spectrophotometer

1. Simple spectrophotometer
- Visible light
- 380 to 750 nm
- Light is produced by a
tungsten lamp
2. UV-vis spectrophotometer
- Second lamp is used:
DEUTERIUM turns a
visible
light
spectrophotometer into a
UV-visible unit that can
measure from: 190-380
nm
- Available with a variety of
features:
scanning,
multiple cells, intergral
printers,
and
user
interfaces

Light comes from a light


source (tungsten lamp or
delirium)
then
to
a
monochromator then llight will
pass through the sample
solution placed in a cuvette
and its wavelength will be in
the digital display
Types of cuvette
1. Glass cuvette
2. Plastiic cuvette

Types of Spectrophotometers

ABSORBANCE AND
CONCENTRATION OF
ABSORBING MOLECULE
Absorbance vs Transmittance
Absorbance: The amount of
light that can be absorbed
Transmittance: The amount
of light that passes through
the solution as it is not
absorbed
Absorbance rather than the
transmittance is most useful
in spectrophotometry
If no light is absorbed
Absorbance = 0
Transmittance = 100%
Each unit in absorbance
corresponds with an order of
magnitude in the fraction of
light transmitted
A=1, 10% of the light is
transmitted (0.10) 90% is
absorbed
A=2, 1% of the light is
transmitted and 99% is
absorbed

A=3, 0,1% of light


transmitted and 99,9%
absorbed

is
is

r= Pearsons correlation
coefficient/correlation coefficient
r= value should be near 1 or 1 for
the graph to be a linear graph

Formula:
Transmittance, T = P / P0
% Transmittance, %T = 100 T
Absorbance,

A = log10 P0 / P
A = log10 1 / T
A = log10 100 / %T
A = 2 - log10 %T
What is the relation of
Concentration
and
Absorbance?
Explained by the Beers Law
Beers Law
A=ebc
Where A is absorbance (no units,
since A
=
log10 P0 /
P)
e is the molar absorbtivity with units
of
L
mol-1 cm-1
b is the path length of the sample - that is, the path length of the
cuvette in which the sample is contained. We will express this
measurement

in

centimetres.

c is the concentration of the compound in solution, expressed in


mol L-

An
increase
in
the
absorbance will also lead to
an
increase
in
the
concentration
(direct
relationship)

Linear Regression
y=mx + b

1. Why was Bromophenol blue


(BPB)
used
in
the
experiment?
BPB was used in the
experiment as an indicator
Can be used as a pH
indicator, color marker or dye
2. Why was vortex used?
The vortex was used to mix
ALL the components of the
solution
3. What
is
the
accurate
absorbance
of
Bromophenol blue?

ACCURACY VS PRECISION

Accuracy
How close the measured
value is to the actual value or
true value
Precision
How close the value of the
measurement is close to the
other measured values by the
group

Analytical Balance
Great precision in quantitative
chemical analysis
Can give up to 4 decimal
places (0.0000g)
Water: 1:1 ratio with mL to g
DENSITY of water:1
Formulas used:
Computing for Concentration (c2)
C1V1=C2V2
Bromophenol blue: 1.25g/mL