Lab 1 (Reagents) Flashcards
What is the purpose of double digestion of plasmids?
This step linearizes the plasmids pGEX-GST and pcDNA3-4EBP1, creating compatible ends for ligation.
BamHI & XhoI
Restriction enzymes that cut at specific DNA sequences, leaving compatible overhangs.
10X Reaction buffer
Provides the optimal pH and salt concentration for enzyme activity.
H20
Dilutes the reaction components to the appropriate concentration.
Alkaline phosphatase (AP)
Removes the phosphate groups from the ends of the linearized pGEX-GST vector, preventing self-ligation.
What is the purpose of the Agarose Gel Electrophoresis?
Separates DNA fragments based on size, allowing visualization and isolation of the desired fragments.
Agarose gel
A porous matrix that allows DNA to migrate under an electric field.
TBE buffer
Conducts electricity and maintains the pH of the gel
DNA Ladder
A mixture of DNA fragments of known sizes, used as a reference to determine the size of the digested fragments.
Red safe stain
A fluorescent dye that binds to DNA, allowing visualization under UV light.
What is the purpose of DNA gel purification?
Isolates the desired DNA fragments (linearized pGEX-GST and 4EBP1 insert) from the agarose gel.
GEL buffer
Solubilizes the agarose gel, releasing the DNA fragments.
Elution Buffer
Releases the purified DNA from the column.
Wash Buffer and W1 Buffer
Used to wash away impurities during the purification process.
Purpose of Ligation
Joins the 4EBP1 insert into the linearized pGEX-GST vector, creating the recombinant plasmid pGEX-GST-4EBP1.
T4 DNA Ligase
An enzyme that catalyzes the formation of phosphodiester bonds between the DNA fragments.
10X T4 Ligation Buffer
Provides the optimal pH and salt concentration for ligase activity.
Purpose of ‘Transformation’
Introduces the recombinant plasmid into competent E. coli DH5a bacteria, which will replicate the plasmid.
Competent E. coli DH5α cells
Bacteria treated to make them capable of taking up DNA.
LB Broth (Lennox) and CaCl2 solution
Used to prepare competent bacteria.
Sterile LB broth
Provide nutrients for bacterial growth.
LB agar plates containing ampicillin
A selective medium that allows only bacteria containing the recombinant plasmid (which carries the ampicillin resistant gene) to grow.
Purpose of Induction of Protein Expression
Induces the expression of the GST-4E-BP1 fusion protein in E.coli BL21 Bacteria.
Terrific Broth (TB) medium plus ampicillin
Provides nutrients for bacterial growth and selects for bacteria containing the pGEX-GST-4EBP1 plasmid.
IPTG
An inducer that binds to the lac repressor, allowing transcription of the gene encoding the GST-4E-BP1 fusion protein.
What is the purpose of cell lysis and crude lysate preparation?
Breaks open the bacteria to release the GST-4E-BP1 protein.
Lysis buffer (containing Tris-HCl, NaCl, EDTA, Triton X-100, lysozyme, DTT, and protease inhibitors)
Breaks down the bacterial cell wall and membrane, releasing the protein.
Sonication
Uses high-frequency sound waves to further break open the cells.
Affinity Purification
Specifically isolates the GST-tagged protein using glutathione-sepharose beads
Glutathione-Sepharose beads
Beads coated with glutathione, which binds to the GST tag on the protein.
PBS
Used to wash the beads and remove unbound proteins.
Glutathione elution buffer
Releases the GST-tagged protein from the beads
Dialysis
Removes the elution buffer and exchanges it with a storage buffer.
Buffer A (containing Tris-HCl, KCl, EDTA, DTT, and glycerol
Provides a suitable environment for protein storage.
How is protein concentration determined?
Determines the concentration of purified protein using the Bradford assay.
Bradford reagent
A dye that binds to proteins, producing a blue color proportional to the protein concentration.
Bovine serum albumin (BSA)
A standard protein used to create a standard curve for the Bradford assay.
SDS-PAGE analysis
Visualizes and estimates the molecular weight of the purified protein.
SDS-PAGE Gel
A polyacrylamide gel that separates proteins based on size under an electric field.
SDS Sample buffer
Denatures and coats proteins with SDS, giving them a uniform charge.
Coomassie Blue Stain
Stains proteins, allowing visualization.
Destaining solution
Removes excess stain from the gel
In Vitro Translation using Rabbit Reticulocyte Lysate
Synthesizes luciferase protein from its mRNA using a cell-free system.
Rabbit Reticulocyte Lysate (RRL)
Contains all the necessary components for protein synthesis, including ribosomes, tRNAs, amino acids, and translation factors.
Luciferase mRNA
The template for protein synthesis
Amino acid mixture
Provide the building blocks for protein synthesis.
KCl
Provides the optimal salt concentration for translation.
Inhibition of Translation.
Tests the effect of GST-4E-BP1 and cycloheximide on translation.
GST-4E-BP1: A known inhibitor of cap-dependent translation.
Cycloheximide: A general inhibitor of protein synthesis.
What is the purpose of the Luciferase assay?
Measures the amount of luciferase protein synthesized, which reflects the efficiency of translation.
Luciferase assay reagent
Contains luciferin, the substrate for luciferase, and other components that generate light in the presence of luciferase.
Reporter Lysis Buffer
Lyses the cells and releases the luciferase protein.
