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71 Cards in this Set
- Front
- Back
PCR components
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DNA template, primers, taq polymerase, dNTPs, buffer, divalent cations, double-distilled water
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dNTPs
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Deoxyribonucleotide triphosphates: DNA monomers
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Taq buffer
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for stable salt concentration and pH for PCR
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Divalent cations
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usually MgCl2, enzyme cofactors required for strand extension by Taq polymerase
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Thermal cycler temperatures for PCR
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94˚ for 3 min
30 to 40 cycles of: 94˚ for 30 sec 56-64˚ for 30 sec 72˚ for 1-3 min then 72˚ for 5 min 10˚ indefinitely |
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agarose is....
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A polysaccharide purified from agar, which comes from seaweed.
A disacchardie composed of galactose and galactopyranose |
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draw agarose
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(blank) look up lab 2
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the agarose matrices serve as ______
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molecular sieves
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markers
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DNA fragments of know lengths
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how do we see DNA in the gel?
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ethidium bromide
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loading dye
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EDTA, glycerol, tracking dyes
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EDTA
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in loading dye, chelates with cation cofactors of DNAases to protect DNA
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tracking dyes
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bromophenol blue or xylene cyanol
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the name of our gene
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TvRad51
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the name of our vector
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pBAD-TOPO
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Three important features of plasmid vectors
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MCS, ORI, selectable marker
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MCS
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multiple cloning site or polylinker
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ORI
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origin of replication, allows plasmid to replicate independently of host chromosomal DNA
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Selectable marker
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resistance to specific antibiotic, in our case ampicillin
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what is special about pBAD-TOPO that makes it awesome for inserting PCR product into?
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Taq polymerase makes sticky A on 3' ends of PCR product, and the pBAD-TOPO vector has overhanging Ts.
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what is the purpose of topoisomerase in our ligation?
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topoisomerase catalyzes formation of new phosphodiester linkages to seal the gaps between the cloning vector and the insert
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definition of transformation
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genetic alteration of a cell as a result of the uptake of foreign genetic material
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how do we make cells chemically competent?
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soak bacteria in divalent cations such as Ca+2
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3 stages of transformation
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Incubation: DNA added to comp cells (in cation solution), at 0˚C
Heat shock: 42˚C, then returned to 0˚C: creates thermal imbalance to create a draft that sweeps plasmid into the cell Recovery: LB broth, incubation at 37˚C, time to recover and express resistance markers |
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what is the purpose of cation solution in incubation step of transformation?
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they neutralize phosphates on DNA and phospholipids of cell membrane to allow DNA to get to the cell membrane
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disadvantages of electroporation
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requires more cells and special instrumentation (more efficient though)
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agarose gel is made of ______
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1% agarose, running buffer (0.5X TBE), and ethidium bromide
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TBE = running buffer
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Tris buffer (for slightly basic soln)
Boric acid gives ions for current EDTA chelates divalent cation cofactors of DNAases |
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the color of the positive pole is _____
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red
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components mixed for TOPO ligation
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PCR product, salt solution, and pBAD-TOPO vector
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how does topoisomerase come in the pBAD-TOPO vector?
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topoisomerase is attached at each end of the linear vector to link PCR product with 3' sticky As to the vector's 3' sticky Ts
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Buffer P1
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Tris-HCl, EDTA, RNase, lysozyme (digests cell wall)
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Buffer P2
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alkaline lysis solution:
SDS (disrupts membranes and denatures macromolecules) NaOH also denatures macromolecules (increased viscosity of solution) |
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Buffer N3 (key step)
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potassium acetate, pH 5.5
-renatures macromolecules non-specifically -high salts help DNA bind to silica membrane |
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silica matrix
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add supernatant from previous miniprep steps so only plasmid DNA is added and binds to column
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Buffer PE
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ethanol to remove salts from the spin column
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Buffer EB
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pH 8.5, elutes DNA with low salt concentration and basic conditions
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EcoR1 restriction site
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GAA---
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EcoRV restriction site
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GAT---
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# of base pairs in pBAD vector
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4 Kb (with EcoRV site about 900 bp before promoter)
4126 (with EcoRV site 950 bp before promoter) |
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# of base pairs in TvRad51 gene
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990 (with EcoRV site about 100 bp in)
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what features does pBAD have that make it easy to get proteins out of bacteria?
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- inducible bacterial promoter
- 6xHis tag - V5 epitope tag |
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what are the characteristics of ideal primers?
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- 20-25 bp
- 50-60% G-C - 3' ends not complementary - forward and reverse have same annealing temperatures - no tandem repeats - no long stretches of same nucleotide |
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How are melting and annealing temperatures related?
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annealing temperature is about 3˚C below the melting temperature
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how is melting temperature calculated?
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4(G+C) + 2(A+T)
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what are examples of protein tags?
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6xhis, FLAG (made up)
myc, HA (derived from other proteins) thioredoxin is a small protein that increases stability of the target protein |
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we purified our protein using a technique called_________
with magnetic beads with ____ ions. ____ ions could also be used. |
affinity chromatography
nickel cobalt |
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how do we elute our proteins from the magnetic beads?
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excess imidazole competes with the 6xHis-tagged protein for nickel, displacing it
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what can cause problems with purifying protein, and what do we have to do to solve the problem?
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If the protein is unhappy in bacteria or made as such high levels that it clumps together, then you have to exchange the native condition buffer for denaturing conditions
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Steps of affinity chromatography
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- cell lysis reagent (buffer and detergents)
- DNase I (removes viscosity) - MagneHis Ni particles - remove supernatant - Binding/Wash buffer - remove supernatant, repeat wash and removal - Elution buffer (500 mM imidazole) - save supernatant |
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SDS-PAGE stands for....
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sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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what are 3 properties of a protein gel that make it more problematic than for DNA?
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- 2˚, 3˚, 4˚ structures
- already differently charged - proteins must enter gel at the same time |
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how do we solve the problem of protein 2˚, 3˚ and 4˚ structure in running a gel?
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- SDS (denatures)
- DTT (dithiothreitol, breaks disulfide bridges) - boiling (increases rate of denaturation) |
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how do we give the proteins a uniform charge to run through a gel?
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each SDS molecule associates with about 2 amino acids giving a uniform negative charge proportional to the protein's molecular weight
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draw an acrylamine monomer
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H2C=C-CO-NH2
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polyacrylamide gels are different from agarose gels because....
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- more resolving power
- vertical - harder to pour and more expensive - harder to purify DNA fragments from |
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what happens in a stacking gel?
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Laemmli's invention:
- gel contains chloride ions - running buffer has glycine ions - they create "ion sandwich" - only 4% acrylamine, so proteins all migrate together regardless of size - pH 6.8: acidic to keep glycine ions behind proteins |
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what happens when proteins enter the resolving gel?
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- pH of 8 makes glycine negatively charged so it speeds ahead of proteins
- proteins now migrate according to size |
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what is the dye used to visualize all proteins after SDS-PAGE?
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Coomassie Brilliant Blue to stain the entire gel
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What helps proteins stick to the nitrocellulose membrane?
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methanol
(and the membrane's very high affinity for amino acids) |
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what is in the transfer buffer for a Western?
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methanol to help proteins stick to nitrocellulose membrane
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what do we do after running the Western transfer for 1 h?
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submerge the cassette in blocking buffer and incubate at 4˚C overnight
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antigen stands for _______ and the parts of antigens where antibodies bind are called ______
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antibody generator
epitopes |
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where do we get primary antibodies from? Which animal is ours from?
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rabbits, mice, goats, sheep, or horses
RABBITS |
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what ways can we label secondary antibodies?
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fluorescence
radioactivity enzyme like HRP or alkaline phosphatase |
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what is the name of our secondary antibody?
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goat Anti-Rabbit
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how do you see AP-labeled antibodies?
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Western Blue substrate contains BCIP and NBT that alkaline phosphatase turns purple
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What solution is the primary antibody in when we add it to our nitrocellulose membrane to do a Western blot?
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TBST: 0.1% Tween-20 in 1x Tris buffered saline solution
(or 5 mL Blocking buffer = 5% milk in TBST) |
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What are the steps to Western blotting after the nitrocellulose membrane has our protein on it?
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Blocking solution (buffer + 5% powdered milk)
1˚ antibody in TBST (60 min) wash with TBST x 3 2˚ antibody in TBST (60 min) wash with TBST x 3 wash with TBS to remove Tween-20 Western Blue substrate for AP |
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What is Tween-20?
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A detergent that interferes with non-specific binding of antibodies to random proteins or to the membrane itself
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What does TBST contain?
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TBS (Tris buffered saline soln)
Tween-20 |