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Bioc320 > Lab 1 (Reagents) > Flashcards

Lab 1 (Reagents) Flashcards

1
Q

What is the purpose of double digestion of plasmids?

A

This step linearizes the plasmids pGEX-GST and pcDNA3-4EBP1, creating compatible ends for ligation.

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2
Q

BamHI & XhoI

A

Restriction enzymes that cut at specific DNA sequences, leaving compatible overhangs.

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3
Q

10X Reaction buffer

A

Provides the optimal pH and salt concentration for enzyme activity.

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4
Q

H20

A

Dilutes the reaction components to the appropriate concentration.

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5
Q

Alkaline phosphatase (AP)

A

Removes the phosphate groups from the ends of the linearized pGEX-GST vector, preventing self-ligation.

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6
Q

What is the purpose of the Agarose Gel Electrophoresis?

A

Separates DNA fragments based on size, allowing visualization and isolation of the desired fragments.

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7
Q

Agarose gel

A

A porous matrix that allows DNA to migrate under an electric field.

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8
Q

TBE buffer

A

Conducts electricity and maintains the pH of the gel

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9
Q

DNA Ladder

A

A mixture of DNA fragments of known sizes, used as a reference to determine the size of the digested fragments.

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10
Q

Red safe stain

A

A fluorescent dye that binds to DNA, allowing visualization under UV light.

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11
Q

What is the purpose of DNA gel purification?

A

Isolates the desired DNA fragments (linearized pGEX-GST and 4EBP1 insert) from the agarose gel.

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12
Q

GEL buffer

A

Solubilizes the agarose gel, releasing the DNA fragments.

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13
Q

Elution Buffer

A

Releases the purified DNA from the column.

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14
Q

Wash Buffer and W1 Buffer

A

Used to wash away impurities during the purification process.

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15
Q

Purpose of Ligation

A

Joins the 4EBP1 insert into the linearized pGEX-GST vector, creating the recombinant plasmid pGEX-GST-4EBP1.

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16
Q

T4 DNA Ligase

A

An enzyme that catalyzes the formation of phosphodiester bonds between the DNA fragments.

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17
Q

10X T4 Ligation Buffer

A

Provides the optimal pH and salt concentration for ligase activity.

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18
Q

Purpose of ‘Transformation’

A

Introduces the recombinant plasmid into competent E. coli DH5a bacteria, which will replicate the plasmid.

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19
Q

Competent E. coli DH5α cells

A

Bacteria treated to make them capable of taking up DNA.

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20
Q

LB Broth (Lennox) and CaCl2 solution

A

Used to prepare competent bacteria.

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21
Q

Sterile LB broth

A

Provide nutrients for bacterial growth.

22
Q

LB agar plates containing ampicillin

A

A selective medium that allows only bacteria containing the recombinant plasmid (which carries the ampicillin resistant gene) to grow.

23
Q

Purpose of Induction of Protein Expression

A

Induces the expression of the GST-4E-BP1 fusion protein in E.coli BL21 Bacteria.

24
Q

Terrific Broth (TB) medium plus ampicillin

A

Provides nutrients for bacterial growth and selects for bacteria containing the pGEX-GST-4EBP1 plasmid.

25
Q

IPTG

A

An inducer that binds to the lac repressor, allowing transcription of the gene encoding the GST-4E-BP1 fusion protein.

26
Q

What is the purpose of cell lysis and crude lysate preparation?

A

Breaks open the bacteria to release the GST-4E-BP1 protein.

27
Q

Lysis buffer (containing Tris-HCl, NaCl, EDTA, Triton X-100, lysozyme, DTT, and protease inhibitors)

A

Breaks down the bacterial cell wall and membrane, releasing the protein.

28
Q

Sonication

A

Uses high-frequency sound waves to further break open the cells.

29
Q

Affinity Purification

A

Specifically isolates the GST-tagged protein using glutathione-sepharose beads

30
Q

Glutathione-Sepharose beads

A

Beads coated with glutathione, which binds to the GST tag on the protein.

31
Q

PBS

A

Used to wash the beads and remove unbound proteins.

32
Q

Glutathione elution buffer

A

Releases the GST-tagged protein from the beads

33
Q

Dialysis

A

Removes the elution buffer and exchanges it with a storage buffer.

34
Q

Buffer A (containing Tris-HCl, KCl, EDTA, DTT, and glycerol

A

Provides a suitable environment for protein storage.

35
Q

How is protein concentration determined?

A

Determines the concentration of purified protein using the Bradford assay.

36
Q

Bradford reagent

A

A dye that binds to proteins, producing a blue color proportional to the protein concentration.

37
Q

Bovine serum albumin (BSA)

A

A standard protein used to create a standard curve for the Bradford assay.

38
Q

SDS-PAGE analysis

A

Visualizes and estimates the molecular weight of the purified protein.

39
Q

SDS-PAGE Gel

A

A polyacrylamide gel that separates proteins based on size under an electric field.

40
Q

SDS Sample buffer

A

Denatures and coats proteins with SDS, giving them a uniform charge.

41
Q

Coomassie Blue Stain

A

Stains proteins, allowing visualization.

42
Q

Destaining solution

A

Removes excess stain from the gel

43
Q

In Vitro Translation using Rabbit Reticulocyte Lysate

A

Synthesizes luciferase protein from its mRNA using a cell-free system.

44
Q

Rabbit Reticulocyte Lysate (RRL)

A

Contains all the necessary components for protein synthesis, including ribosomes, tRNAs, amino acids, and translation factors.

45
Q

Luciferase mRNA

A

The template for protein synthesis

46
Q

Amino acid mixture

A

Provide the building blocks for protein synthesis.

47
Q

KCl

A

Provides the optimal salt concentration for translation.

48
Q

Inhibition of Translation.

A

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.

49
Q

What is the purpose of the Luciferase assay?

A

Measures the amount of luciferase protein synthesized, which reflects the efficiency of translation.

50
Q

Luciferase assay reagent

A

Contains luciferin, the substrate for luciferase, and other components that generate light in the presence of luciferase.

51
Q

Reporter Lysis Buffer

A

Lyses the cells and releases the luciferase protein.

Bioc320 (6 decks)

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