BMB 212 LAB exam 2

Which techniques were performed during the BMB lab?

Plasmid purification using a mini prep procedure and using absorbance (A260/A280) to analyze DNA concentration and purity.

Which techniques were performed before the lab session?

Bacterial transformation, cloning GFP using restriction enzymes and DNA ligase, and DNA Purity (A260/A280) analysis.

What does an A260/A280 ratio indicate?

DNA purity, specifically identifying protein or RNA contamination.

What is the ideal A260/A280 ratio for pure DNA?

~1.8

What does an A260/A280 ratio below 1.8 indicate?

Protein contamination.

What does an A260/A280 ratio above 2.0 indicate?

RNA contamination.

What is an XbaI site?

A restriction enzyme recognition site where DNA is cut.

What is the function of the T7 promoter?

It controls gene expression in the plasmid.

What is KanR?

An antibiotic resistance gene providing kanamycin resistance.

What is the function of the ori sequence?

It is the origin of replication, allowing the plasmid to replicate within bacteria.

What is the purpose of a 6xHis tag?

It is a protein tag used for the purification of expressed protein.

What are the standard units for reporting DNA concentration?

ng/µL

What is the formula for calculating DNA yield?

Yield = concentration (ng/µL) × volume (µL)

What is the formula for calculating the volume needed for a specific DNA mass?

Volume = desired mass ÷ concentration

Which enzyme cuts both plasmid and insert DNA?

Restriction enzymes.

Which enzyme is used to join DNA fragments together?

DNA ligase.

What gene is inserted in this experiment?

GFP (green fluorescent protein).

What is the purpose of antibiotic selection in cloning?

To ensure that only bacteria containing the plasmid survive.

What antibiotic is used in this system?

Kanamycin.

What are the two final bacterial populations expected after cloning?

Bacteria with empty plasmids and bacteria with recombinant plasmids (GFP inserted).

What does an A260/A280 ratio of 1.81 indicate?

Pure DNA with minimal contamination.

What factors can cause a failed purification resulting in a bad A260/A280 ratio?

Protein contamination (low ratio), RNA contamination (high ratio), or poor washing during the mini prep.

Recombinant DNA

DNA formed by combining DNA from different sources

Purpose of recombinant DNA

To produce proteins of interest

Restriction enzymes

Proteins that cut DNA at specific sequences

Sticky ends

Overhanging DNA ends that can base-pair

Blunt ends

DNA ends with no overhang

DNA ligase

Enzyme that joins DNA fragments together

Transformation

Uptake of plasmid DNA by bacteria

Selectable marker

Gene (e.g., antibiotic resistance) used to identify transformed cells

Origin of replication (ori)

Site where DNA replication begins

Promoter

DNA sequence where transcription begins

Plasmid map

Diagram showing locations of genes and restriction sites

Mini prep

Method used to isolate plasmid DNA from bacteria

Alkaline lysis

Technique using SDS and NaOH to break cells and denature DNA

Why plasmid DNA renatures

It is circular and supercoiled

Spin column

Used to purify DNA by binding to silica

A260

Absorbance used to measure nucleic acid concentration

A280

Absorbance used to measure protein contamination

A260/A280 ≈ 1.8

Pure DNA

A260/A280 < 1.8

Protein contamination

A260/A280 > 2.0

RNA contamination

Supercoiled DNA

Migrates fastest on gel

Nicked (open circular) DNA

Migrates slowest

Linear DNA

Migrates at intermediate speed

Restriction digest

Cutting DNA with restriction enzymes

Agarose gel electrophoresis

Separates DNA by size

DNA charge

Negative (moves toward positive electrode)

Smaller DNA fragments

Travel farther on gel

Agarose %

Higher % = better separation of small fragments

DNA ladder

Standard used to estimate fragment size

Gene expression

DNA → RNA → Protein

Transcription

DNA → mRNA

Translation

mRNA → protein

T7 promoter

Strong promoter for high protein expression

Lac operon

Controls gene expression

IPTG

Inducer that removes repressor

Affinity purification

Isolates proteins based on binding

His-tag

سلسلة histidine residues added to protein

Ni-NTA column

Resin that binds His-tagged proteins

Imidazole

Competes with histidine to elute protein

SDS-PAGE

Separates proteins by size

SDS

Denatures proteins and gives negative charge

Reducing agent (β-mercaptoethanol)

Breaks disulfide bonds

Saponification

Hydrolysis of triglycerides to form soap

Saturated fats

No double bonds, pack tightly

Unsaturated fats

Double bonds, kinked structure