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biochemistry
lab midterm
75
Biology
Undergraduate 4
03/06/2009

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Term
what antibiotic is similar to
Definition
carbenicillin
Term
what is ampicillins mode of action
Definition
inhibit transpeptidase. It is most effective in log phase
Term
What is the mechanism for resistance against ampicillin
Definition
Beta lactamase
Term
What is kanamycins mode of action
Definition
IT binds 3 ribosomal proteins and specific bases in 30s ribosomal units. Its an aminoglycoside antibiotic. Polycations diffuse through porin channels in outrer membranes of gram-negative bacteria. Engergy driven transport via negative membrane potential into cytoplasm. They decrease protein synthesis and increase transcriptional errors
Term
What is the mechanism of resistance against kanamycin
Definition
Aminophosphotransferases. THey transfer gamma phopshate of ATP to OH in 3' position of pseudosaccharide.
Term
How does tetracycline work
Definition
They bind to a single site on 30 ribosomal subunit- preventing binding of aminacyl-tRNA to acceptor site. Protein synthesis is blocked.
Term
What is the mechanism of Resistance against tetracycline
Definition
Antiporter Tet proteins. found in inner membrane. They exchange a proton for intracellular tetracycline-metal complexes against a concentration gradient. 5 similar genes for tetracycline efflux.
Term
what is the formula for ion movement in an electric field
Definition
V= velocity of a charged particle moving in an electric field.
V= (field strength)(charge on particle)/((size and shape of molecule)(viscosity of solution))
Term
With small protein MWs should you have a higher or lower % of arylamide
Definition
higher
Term
what does SDS stand for
Definition
sodium dodecyl sulphate
Term
what are the additional denaturing agents?
Definition
1. Heat
2. Beta mercaptoethanol (BME) and Dithiothreitol (DTT)
3. EDTA
Term
what do BME and DTT do
Definition
they break the disulfide bonds in proteins
Term
what does EDTA do
Definition
Bind metal ion cofactors, keeps plasmids and proteins from aggregating.
Term
What are the ways that proteins can be visualized on gel?
Definition
1. coomassie blue staining (100ng/ band)
2. Silver nitrate staining (1-10ng/ band)
3. SYPRO
Term
what are some of the application of SDS
Definition
Determine protein purity
Determine molecular weight of proteins
Term
Describe basic steps of Western blotting
Definition
1. SDS-polyacrylamide gel electrophoresis
2. Transfer proteins to nitrocellulose membranes
3. use specific antibody to identify target protein.
Term
What does ELISA stand for
Definition
Enzyme-Linked immunosorbent Assays
Term
Describe basics of Agarose gel
Definition
A highly purified polysaccharide derived from agar
concentration of agarose can be varied from 0.5 to 3%.
the higher the concentration, the smaller the pores will be in the solidified gel.
Term
ethidium bromide
Definition
can be used to visualize DNA or proteins
Intercatalating agent used as fluorescent tag.
Term
Define operon
Definition
a coordinated unit of gene expression. Include a cluster of structural genes which envode products involved in a set of related processes plus regulatory elements which control their expression. 2-6 genes norm but can be 20 +
Term
how many binding domains does the lac repressor have
Definition
4
Term
what are the levels of cAMP like in the presence of glucose
Definition
low
Term
What is formed when lacZ alpha and omega come together?
Definition
beta galactosidase
Term
what does lacZ alpha do?
Definition
encodes amino- terminal fragment
Term
what does lacZ omega do.
Definition
encodes carboxy terminal fragment.
Term
what is the inducer of Beta galactosidase
Definition
IPTG
Term
What 2 components are required for T7 promoter control
Definition
1. plasmid with T7 promoter upstream of gene to be expressed.
2. Bacteriophage T7 RNA polymerase
a. Only recognizes T7 promoter
b. Can be on vector or gene inserted into E coli chromosome.
Term
what does IPTG stand for
Definition
isopropylthiogalactoside
Term
what types of cells express the proteins
Definition
tuner cells
Term
Does pLacI have antibiotic resistance
Definition
yes with the cat gene
Term
what are the 4 antiobiotics we talked about
Definition
1. Ampicillin (Carbenicillin)
2.Chloramphenicol
3. Kanamycins
4. Tetracycline
Term
Which antibiotics act on ribosomes
Definition
all but ampicllin and carbicillin
Term
What does chloramphenicol act on
Definition
it inhibits the peptidly transferase on 70s ribosomes. causes decrease protein synthesis and decreased hot DNA synthesis
Term
What is the mechanism of resistance against chloramphenicol
Definition
the chloramphenicol acetyltransferase (cat gene)
Term
Which carbon isotopes are unstable
Definition
carbon 14
Term
Carbon 14 decays into Nitrogen 14. what gets emitted
Definition
a beta negative particle
Term
is the geiger counter qualitative or quantitative
Definition
qualitative.
Term
what method is quantitative for measurement of radioactivity
Definition
liquid scintillation counting
Term
what are some of the widely used primary and secondary fluors
Definition
PPO, bis MSB, Dimethyl-POPOP
Term
counting efficiency
Definition
cpm=dpm times efficiency of countring
Term
Describe some basic characteristic of plasmid DNA
Definition
1. dependent of host enzymes for replication and transcription
2. no coat protein
3. maintain stable copy numbers
4.
Term
define replicon
Definition
a DNA molecule, RNA molecule, or a region that replicates from a single origin of replication. The smallest piece of plasmid DNA able to replicate autonomously and maintain copy number.
Term
what does maintaining copy number depend on?
Definition
frequency of initiation of plasmid DNA synthesis
Term
Stringent
Definition
(referes to replicon) Plamsid encoded protein required for initiation of synthesis. Cannon be amplified by decreased protein synthesis.
Term
Relaxed
Definition
(refers to replicon) RNAII required for initiation of synthesis. do not require protein syntheis. yield increased by decrease cell protein synthesis.
Term
describe the phases of virusus
Definition
the lysogenic phase is where the virus dna is just chillin. In the lytic phase the dna is overexpressed untill cell lyses
Term
what percentage of normal genome can get packaged in a bacteriophage
Definition
75-105%
Term
what are cosmids
Definition
the dna that can get brough to a cell through a bacteriopahge
Term
what are shuttle vectors
Definition
plasmids transferring genes between two organisms. Prokaryote and eukaryote. Unique origins of replication for each cell type. selectable markers for each cell type.ampicillinclone eukaryotic genes in prokaryote, but express in eukaryotes.
Term
what is BAC
Definition
bacterial artificial chromosomes. versions of E. coli F factor. inserts are as long as 300 kb. selectable markers. helicase to facilitate DNA replication. 3 loci for accurate positioning of low copy number plasmids to daughter cells.
Term
what is a YAC
Definition
yeast artificial chromosome. centromere. autonomoulsy replicating sequence. pair of telomeres. selectable markers cloning site. inserts as large as 1000 kb. transferred into higher organisms
Term
Why is DNA more stable than RNA
Definition
because the Thymidine has a methyl group on its ring
Term
who invinted PCR
Definition
karry mullis
Term
what are the components of PCR reaction
Definition
1. buffer(containing Mg++) to keep ph consistant.
2. Template DNA
3. 2 primers to flank the copy to be replicated.
4. dNTPS
5. taq polymerase
Term
How do you calculate the theoretical yield of PCRq
Definition
(2 raised the the n power) times the starting number of copies
Term
what are 4 application of PCR
Definition
1. addition of a restriction site
2. DNA deletion
3. Site specific mutagenesis
4. random mutagenesis
Term
what is sloppy mutagenesis
Definition
different ratios of dNTPs. Mn++ instead of Mg. low fidelity enzyme
Term
what does reverse transcriptase do
Definition
Transcribes mRNA into ds cDNA
Term
what is the differrence between restriction endonucleases and modification enzymes
Definition
they both are defense systems, but the endonuclease cleaves while the modification enzyme methylates.
Term
what are type 3 endonucleases
Definition
cut at specific sites and dissociate.
Term
what are type 1 endonucleases
Definition
cut at random sites when DNA loops back to bound enzyme
Term
type 2 endonucleases
Definition
recognize specific sequences and methylate
Term
what are the functions of kinases
Definition
to transfer gamma phosphate of ATP to 5'-OH in DNA or RNA
Term
phosphatases
Definition
remove phosphate group from 5' end and leave OH group
Term
What do you mix the agarose with to get the gel
Definition
TAE
Term
what are important factors in choosing a good buffer
Definition
1. solubility
2. pH of enzyme
3. temperature
4. charge
Term
what is the difference between RNA and DNA
Definition
the DNA does not have a hydroxy group at the 2' posisition
Term
how does iptg induce expression
Definition
by binding to the lac repressor
Term
what was included in both plates (transformattion)
Definition
ampicillin and glucose
Term
What is the funtion of TEMED in the polymerization of polyacrylamide gels
Definition
to catalyze the transfer of the radical electron from sulfate to acrylamide
Term
In the mini prep of the plasmid, what were the components and what were their functions
Definition
1. resuspension buffer (glucose and Tris, and EDTA) the glucose balances the osmotic pressure. the Tris stabilizes the pH. The EDTA bind the metal cofactors
2. Lysis solution (sds and NAOH) the NaOH bseparates the ds DNA to form ssDNA adding in the break down of cell bembrane. the SDS denatures the proeins and destroys the cell membrane
3. Neutralizing buffer (KOAc and acetic acid)the acetic acid neutralizes the basic conditions. the KOAc allows for base pairing to reoccur by pulling water from DNA strands aiding in precipitate to form with isoproanol.
Term
what are the products produced when beta galactosidase cleaves lactose
Definition
glucose and galactose.
Term
what blue protein gets made from xgal
Definition
5 br 4 Cl indole
Term
Electroporation use what conditions
Definition
2500 v 6 ms
Term
what is the enzyme atached to the goat enzyme
Definition
alchaline phosphatase
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