EXAM

Question 1

What are the two E.coli strains used in the lab?

Answer 1

EMG 26 K-12 Lac-(i+z-y+)
EMG 9 K-12 lac (i-z+y+)

Question 2

Which strain makes the galactosidase enzyme?

Answer 2

EMG 9 produces it because it has the lac z gene.

Question 3

how to produce a protein profile based on molecular weight?

Answer 3

SDS-PAGE is used to extract the SDS-PAGE

Question 4

What filter is used in the electrophretic transfer?

Answer 4

Nitrocellullose filter

Question 5

What antibody is used to visualize the beta-galactosidase protein on the nitrocellulose filter?

Answer 5

Using an anti beta-galactosidase monoclonal antibody followed by an anti-IgG alkaline phosphatase conjugate

Question 6

What gene takes lactose as substrate and cleaves it into monosaccharides galactose and glucose?

Answer 6

lac Z

Question 7

What gene encodes lactose permease, a membrane transport protein that transports lactose into cell to be broken down?

Answer 7

Lac Y

Question 8

What gene codes for repressor that binds to the operator and inhibits transcription?

Answer 8

Lac I

Question 9

What gene codes for beta-galactoside transacetylase that added acetyl groups to lactose and other galactose?

Answer 9

Lac A

Question 10

What is CAP?

Answer 10

Catabolite activator protein increase activation and rate of transcription

Question 11

What membrane is used to visuallize thr beta-galactosidase using the anti-bodies?

Answer 11

PVDF membrane for immuno-blotting

Question 12

what are monoclonal antibodies?

Answer 12

they are generated by identical b-cells and have monovalent affinity and recognize the same epitope of the antigen

Question 13

what are polyclonal antibody?

Answer 13

it is heterozgenous and produces by different b-cells in the body. They bind to many different epitopes of a single antigen.

Question 14

What is the role of methanol?

Answer 14

It hydrates the membrane because the membrane is hydrophobic and water will not be able to do anything.

Question 15

What are detergents?

Answer 15

They disrupt the membrane and denature it breaking the protein-protein bond.

Question 16

What does SDS do?

Answer 16

It coats the protein with a negative charge so that they are separated by weight and not charge

Question 17

What is Mercaptoethanol in the lysis buffer?

Answer 17

is a reducing agent that reduces (disrupts) disulphide bridges in proteins, which
is necessary for separation by size.

Question 18

Why is PVDF used for wester blotting?

Answer 18

t immobilizs
proteins, due to its non-specific affinity for amino acids

Question 19

What is the key difference of polyacrilamie and acrylamide?

Answer 19

polyacrylamide is a
polymer and acrylamide is the sub unit used to produce polyacrylamide molecules

Question 20

what is required in a pcr?

Answer 20

DNA polymerase,
 oligonucleotide primers,
 the co-factor Mg2+ (needed for activity of DNA polymerases).
 the four deoxynucleotides, the essential building blocks of nucleic acid molecules

Question 21

what does each PCR cycle consists of?

Answer 21

• denaturation: several minutes at 94-96C

-Annealing: several minutes at 50-65C where the primers are hybridized (H bonding) to complementary seq

-Extension: several minutes at 72C where the polymerase binds and extends a complentary DNA strand from each primer

Question 22

What is DNA barcoding

Answer 22

use info of one or a few gene regions to identify all species

Question 23

what is a positive PCR control

Answer 23

sample that contains a known barcode gene seq

Question 24

what is a negative PCR control

Answer 24

sample that contains water and no DNA no amplification of ay target sew in the sample

Question 25

why does DNA have a negative charge?

Answer 25

because of the phosphodiester bonds

Question 26

what is a primer-dimer?

Answer 26

consists of 2 primer molecules that were hybridized (annealed) to each other bc of complementary bases in their seq

Question 27

why is agarose used for in PCR?

Answer 27

its used for seperation of nucleic acid molecules

Question 28

What is tracking dye?

Answer 28

dna loading dye that is charged and have a low MW. its loaded in each well at the start to monitor the progress of molecule movement on the gel

Question 29

What is RedSafe in PCR?

Answer 29

its a nucleic acid staining solution for detecting nucleic acid in agarose gels and emits a gree fluoresence when bound to DNA or RNA/

Question 30

what are the two common buffers in nucleic acid electrophoresis?

Answer 30

Tris acetate EDTA (TAE) and tris borate EDTA (TBE)

Question 31

what is a TE buffer?

Answer 31

it protects DNA or RNA from degradation

Question 32

what is a chelex?

Answer 32

is a cleating resin that is bound to a Mg2+ and inhibit the activity of enzymes that would degrade DNA

Question 33

what does a lysis buffer have?

Answer 33

contains glycerol, EDTA and 2% mercaptoethanol

Question 34

What does glycerol do in the lysis buffer

Answer 34

Glycerol makes the sample more dense than the sample buffer, so the sample will remain in the
bottom of a well rather than float out

Question 35

What does EDTA do in the lysis buffer

Answer 35

EDTA is a chelating agent and its role in the lysis buffer is to reduce oxidation damage and to
chelate metal ions (and this inhibits some proteases)

Question 36

What is a taq polymerase?

Answer 36

its a thermostable polymerase, can grow at high tempers 67˚C