Term
| what antibiotic is similar to |
|
Definition
|
|
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
|
|
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 |
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|
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. |
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|
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
|
|
Term
|
Definition
|
|
Term
| what are the additional denaturing agents? |
|
Definition
1. Heat
2. Beta mercaptoethanol (BME) and Dithiothreitol (DTT)
3. EDTA |
|
|
Term
|
Definition
| they break the disulfide bonds in proteins |
|
|
Term
|
Definition
| Bind metal ion cofactors, keeps plasmids and proteins from aggregating. |
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|
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
|
Definition
can be used to visualize DNA or proteins
Intercatalating agent used as fluorescent tag. |
|
|
Term
|
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
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|
Term
| what are the levels of cAMP like in the presence of glucose |
|
Definition
|
|
Term
| What is formed when lacZ alpha and omega come together? |
|
Definition
|
|
Term
|
Definition
| encodes amino- terminal fragment |
|
|
Term
|
Definition
| encodes carboxy terminal fragment. |
|
|
Term
| what is the inducer of Beta galactosidase |
|
Definition
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|
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. |
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|
Term
|
Definition
|
|
Term
| what types of cells express the proteins |
|
Definition
|
|
Term
| Does pLacI have antibiotic resistance |
|
Definition
|
|
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
|
|
Term
| Carbon 14 decays into Nitrogen 14. what gets emitted |
|
Definition
|
|
Term
| is the geiger counter qualitative or quantitative |
|
Definition
|
|
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
|
Definition
| cpm=dpm times efficiency of countring |
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|
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
|
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
|
Definition
| (referes to replicon) Plamsid encoded protein required for initiation of synthesis. Cannon be amplified by decreased protein synthesis. |
|
|
Term
|
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
|
|
Term
|
Definition
| the dna that can get brough to a cell through a bacteriopahge |
|
|
Term
|
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
|
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
|
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
|
Definition
|
|
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
|
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
|
Definition
| remove phosphate group from 5' end and leave OH group |
|
|
Term
| What do you mix the agarose with to get the gel |
|
Definition
|
|
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
|
|
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
|
|
Term
| what blue protein gets made from xgal |
|
Definition
|
|
Term
| Electroporation use what conditions |
|
Definition
|
|
Term
| what is the enzyme atached to the goat enzyme |
|
Definition
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