EXAM Flashcards
What are the two E.coli strains used in the lab?
EMG 26 K-12 Lac-(i+z-y+)
EMG 9 K-12 lac (i-z+y+)
Which strain makes the galactosidase enzyme?
EMG 9 produces it because it has the lac z gene.
how to produce a protein profile based on molecular weight?
SDS-PAGE is used to extract the SDS-PAGE
What filter is used in the electrophretic transfer?
Nitrocellullose filter
What antibody is used to visualize the beta-galactosidase protein on the nitrocellulose filter?
Using an anti beta-galactosidase monoclonal antibody followed by an anti-IgG alkaline phosphatase conjugate
What gene takes lactose as substrate and cleaves it into monosaccharides galactose and glucose?
lac Z
What gene encodes lactose permease, a membrane transport protein that transports lactose into cell to be broken down?
Lac Y
What gene codes for repressor that binds to the operator and inhibits transcription?
Lac I
What gene codes for beta-galactoside transacetylase that added acetyl groups to lactose and other galactose?
Lac A
What is CAP?
Catabolite activator protein increase activation and rate of transcription
What membrane is used to visuallize thr beta-galactosidase using the anti-bodies?
PVDF membrane for immuno-blotting
what are monoclonal antibodies?
they are generated by identical b-cells and have monovalent affinity and recognize the same epitope of the antigen
what are polyclonal antibody?
it is heterozgenous and produces by different b-cells in the body. They bind to many different epitopes of a single antigen.
What is the role of methanol?
It hydrates the membrane because the membrane is hydrophobic and water will not be able to do anything.
What are detergents?
They disrupt the membrane and denature it breaking the protein-protein bond.
What does SDS do?
It coats the protein with a negative charge so that they are separated by weight and not charge
What is Mercaptoethanol in the lysis buffer?
is a reducing agent that reduces (disrupts) disulphide bridges in proteins, which
is necessary for separation by size.
Why is PVDF used for wester blotting?
t immobilizs
proteins, due to its non-specific affinity for amino acids
What is the key difference of polyacrilamie and acrylamide?
polyacrylamide is a
polymer and acrylamide is the sub unit used to produce polyacrylamide molecules
what is required in a pcr?
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
what does each PCR cycle consists of?
- 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
What is DNA barcoding
use info of one or a few gene regions to identify all species
what is a positive PCR control
sample that contains a known barcode gene seq
what is a negative PCR control
sample that contains water and no DNA no amplification of ay target sew in the sample
