Lecture 1

Question 1

Define accuracy in pipetting

Answer 1

‐ the degree of agreement of a set of
measurements of a sample to the actual
(true) value of the sample

Question 2

Define precision in pipetting

Answer 2

the degree to which repeated
measurements agree closely with each
other (also called reproducibility or
repeatability)

Question 3

What technique is used to correct pipetting

Answer 3

Pipette held vertically, tip immerse about 1 cm into the liquid

Question 4

What is a spectrophotometer

Answer 4

instrument to measure the absorption or emission of electromagnetic
radiation (or light) by a solution

Question 5

What is the absorption of the spectrophotomer calculated as

Answer 5

T = I/Io
A = log10(1/T)
Beer‐Lambert Equation: A = ε c l

Question 6

What is spectrophotometer used for

Answer 6

• Determine absorption characteristics of a compound*
• Determine concentration of a solution*
• Measure rate of reaction of enzymes

Question 7

What are detected when absorbance is at 280nm

Answer 7

Due to presence of aromatic amino acid
residues within the protein,
e.g. tyrosine and tryptophan

Question 8

How do you calculate the purity of DNA

Answer 8

A260/280 = 0.82 ÷ 0.50 = 1.64
 Pure DNA : A260nm/280nm = 1.8
 RNA contamination: > 1.8
 Protein contamination: <1.8

Question 9

What is Gel electrophoresis

Answer 9

Separation of molecules (e.g. Protein and DNA) through a gel matrix
under electric current, based on size and charge

Question 10

How does Agarose gel electrophoresis work

Answer 10

Separation of molecules based on size and charge
• Negative molecules towards anode
• Positive molecules towards cathode

Separation of DNA molecules
Separation of native proteins: LDH isoenzymes
Detection by UV‐excitation of:
• RedSafeTM DNA staining solution

Question 11

How do you prepare a agarose gel

Answer 11

• melt agarose in buffer
solution
• pour into plastic tray
with comb to generate
wells, cool to set
o agarose/buffer solution
will solidify to form a
semisolid gel
containing pores

Question 12

What does % (w/v) agarose tell us

Answer 12

% agarose determines
size of DNA that can be
separated (‘resolved’)

o high percentage will
‘resolve’ small DNA
fragments

o low percentage will
‘resolve’ larger DNA
fragments

Question 13

What are the ranges of %(w/v) agarose

Answer 13

0. 5, 1 – 30 kbp
1. 7, 0.8 – 12 kbp
2. 0, 0.5 – 10 kbp
3. 2, 0.40 – 7 kbp
4. 5, 200 bp – 3 kbp
5. 0, 50 bp – 2 kbp

Question 14

What are the ways to determine the size of the fragments

Answer 14

• Identify success of digests by
unique bands
• Identify bands of interest
using a calibration curve of
marker DNA of known size

Question 15

What is Vertical acrylamide gel electrophoresis

Answer 15

Analytical method to separate

and visualise proteins

Question 16

What is the use of Vertical acrylamide gel electrophoresis

Answer 16

• determine purity of a
protein preparation (i.e.
efficacy of purification
process)
• determine properties such
as approximate molecular
weight and isoelectric point
(2‐D gels) estimate the
number of proteins within
a mixture