Lab 1-3

What is transformation? How is it done and why?

Transformation: gene transfer into bacteria

How it is done: Introduction of DNA into living cells

Why: Molecular Biology could not exist; Three Main Reasons

1. Propagation of replication-competent DNA, so large amounts can be “grown up” and isolated; large amounts can be purified, analyzed, manipulated further/ used for engineering of other types of cells (ex. Yeast or mammalian cells)

2. Introduced DNA encodes a protein of interest & cells serve as living factories that produce large amounts of desired protein, which can be purified

3. Gene transfer allows properties of DNA to be examined in living cells (ex. Effects of genes present in DNA can be assessed)

What parameters must be considered for maximum efficiency in bacterial transformation?

Parameters: Large number of parameters can affect efficiency of bacterial transformation

1. Bacteria must carry appropriate mutations so foreign DNA is not modified or degraded

2. Bacteria should be actively growing

3. Type of chemical treatment

4. DNA should be intact: circular, closed, supercoiled DNA is the best, linear works poorly

What do you mean by competence? How are cells made competent?

Competence is the ability of bacteria to naturally take up DNA

Cells are made made competent through two treatments: physical & chemical

• Chemical: involves treating cells with ice-cold CaCl2 followed by brief heat shock at 37-42 Celsius (1970)

• Physical: Called “electroporation”, involves exposing E. Coli to brief electric shock (2,500 V for 5 milliseconds) in presence of DNA to be introduced

What are factors that impact transformation success?

• Plasmid concentration: More plasmid = higher chance a cell encounters and takes up DNA during transformation.

• Cell competency: Competent cells have more permeable membranes (chemically or electrically treated), making it easier for DNA to enter.

• Heat shock: A sudden temperature increase creates a temporary imbalance across the membrane, helping DNA move into the cell.

• State of cells: Actively growing (log-phase) cells have more fluid membranes and active transport systems, so they take up DNA more efficiently.

What is a plasmid and what features does it have?

A small circular DNA molecule with an origin of replication and antibiotic resistance genes.

What happens in each step of the transformation protocol- incubation on ice, heat shock and culturing at 37C?

1. Incubation on ice: Slows the cells and stabilizes their membranes while allowing plasmid DNA to attach to the cell surface.

2. Heat shock: A rapid temperature increase creates temporary pores in the membrane, allowing the plasmid DNA to enter the cells.

3. Culturing at 37 °C: Cells recover, begin growing, and express the plasmid genes (like antibiotic resistance), allowing transformed cells to survive and multiply.

What are the steps to bacterial transformation?

• Mixed competent E. coli cells with each unknown plasmid

• Incubated on ice (30 min) → DNA attaches to cells

• Performed heat shock at 42°C (45 sec) → DNA enters cells

• Recovered cells in nutrient broth at 37°C (~1 hour)

• Plated different volumes (100, 250, 500 µL) on:

  • Ampicillin plates

  • Kanamycin plates

What results was observed in bacterial transformation?

• Growth on ampicillin plates only → plasmid = pAMP

• Growth on kanamycin plates only → plasmid = pKAN

• No growth → cells were not transformed

How is selection done for positive clones?

Selection is done using antibiotic-containing plates: only bacteria that successfully took up the plasmid survive b/c the plasmid carries an antibiotic resistance gene

• Amp plate → only cells with ampicillin resistance gene survive

• Kan plate → only cells with kanamycin resistance gene survive

What does the colony size indicate on Amp and Kan plates?

Colony size meaning:

Large colonies → cells transformed early, grew well, strong expression

Small colonies → slower growth, possibly lower plasmid expression or later transformation

No colonies → no transformation occurred

(Kanamycin often gives slower/smaller colonies than ampicillin because it is more stable and acts differently.)

How do the antibiotic selections work? Can other selection markers be used?

Plasmids carry resistance genes that protect bacteria from antibiotics:

• Ampicillin (pAMP):

  • Encodes β-lactamase

  • Breaks down ampicillin → bacteria survive

• Kanamycin (pKAN):

  • Encodes an enzyme that inactivates kanamycin

  • Allows protein synthesis to continue

Other markers include: antibiotic resistance genes (Tetracycline, Chloramphenicol)

What are all steps involved in isolating plasmids from bacterial cells?

Steps (alkaline lysis method):

1. Grow bacterial culture containing plasmid

2. Centrifuge → pellet cells

3. Resuspend pellet (P1)

4. Lyse cells (P2)

5. Neutralize (P3/N3)

6. Centrifuge → separate plasmid from debris

7. Bind DNA to column

8. Wash column

9. Elute purified plasmid DNA

What is the function of each reagent that was used for suspension, lysis, neutralization, binding to column and elution?

Function of each reagent:

• Suspension (P1):

  • Resuspends cells

  • Contains RNase → degrades RNA

  • Helps create uniform solution

• Lysis (P2):

  • Contains NaOH + detergent (SDS)

  • Breaks open cells

  • Denatures DNA, proteins, membranes

• Neutralization (P3/N3):

  • Neutralizes pH

  • Causes chromosomal DNA + proteins to precipitate

  • Plasmid DNA (small, circular) re-anneals and stays in solution

• Binding to column:

  • High salt allows DNA to bind to silica

• Wash:

  • Removes salts, proteins, contaminants

• Elution (solution T):

  • Low salt → DNA releases from column

  • DNA collected in clean tube

How does the Qiagen resin work?

• Qiagen columns contain silica resin (modified silica)

• DNA binds to silica in high-salt conditions

• Contaminants (proteins, RNA, debris) are washed away

• DNA is released when low-salt solution is added

 Key idea: Bind → Wash → Elute

What contaminants can arise in plasmid preps?

• RNA (if RNase is ineffective)

• Proteins

• Genomic (chromosomal) DNA

• Salts and detergents

• Cell debris

How are the quality and quantity of plasmid isolated assessed?

Measured using UV spectrophotometry (NanoDrop)

Quantity: Based on absorbance at 260 nm (A260)

Quality: Determined by A260/A280 ratio

• ~1.8–2.0 = pure DNA

• Lower ratio = protein contamination

• DNA absorbs strongly at 260 nm

What formula is utilized for calculating plasmid content based on its absorbance at 260nm?

DNA concentration = A260 × 50 µg/mL × dilution factor

What does OD260, OD280 and A260/280 ratio mean?

• OD260 (A260): Measures nucleic acids (DNA/RNA)

• OD280 (A280): Measures protein

• A260/A280 ratio:

  • ~1.8–2.0 → pure DNA

  • <1.8 → protein contamination

How does a nanodrop work and what are we measuring when we place nucleic acids in the ports?

NanoDrop:

• Uses UV light absorbance

• Very small sample (~1–2 µL)

• Measures how much light DNA absorbs at 260 nm

• Measures:

  • DNA concentration

  • Purity (via ratios)

Can DNA be measured by alternate ways, besides spectrophotometry?

Other methods to measure DNA:

• Agarose gel electrophoresis (visual estimate)

• Fluorescent dyes (e.g., SYBR Green)

• Qubit fluorometer (more accurate than NanoDrop)

Calculate the plasmid content if A260 is 0.2 and the dilution factor is 10.

DNA concentration = 0.2 × 50 × 10 = 100 µg/mL

What are restriction enzymes and how do they work?

Restriction enzymes (REs):

• Enzymes that cut DNA at specific sequences

How they work:

• Recognize short, palindromic DNA sequences

• Bind DNA and cleave the phosphodiester backbone

Who makes these enzymes and why?

Who makes them & why:

• Produced by bacteria

• Serve as a defense mechanism against foreign DNA (ex. bacteriophages)

• Cut invading DNA to inactivate it

How do REs cut and what kind of ends do they generate?

Cut both DNA strands at specific recognition sites

Types of ends:

• Sticky (cohesive) ends → staggered cuts, single-stranded overhangs

• Blunt ends → straight cuts, no overhang

• Example from lab: EcoRI creates a 5’ overhang

What sequences do EcoRI, BamHI and HindIII recognize and where do they cut?

EcoRI:

• Recognition: GAATTC

• Cut: G↓AATTC

BamHI:

• Recognition: GGATCC

• Cut: G↓GATCC

HindIII:

• Recognition: AAGCTT

• Cut: A↓AGCTT

All cut within specific sequences and generate sticky ends

What is the bacterial restriction/modification system and why is it crucial?

Consists of:

• Restriction enzyme → cuts DNA

• Methylase → protects host DNA

How it works:

• Bacterial DNA is methylated → protected

• Foreign DNA is unmethylated → cut and destroyed

Crucial because:

• Prevents self-DNA degradation

• Protects against viral infection

What do you mean by the terms- cohesive, blunt, overhang?

Cohesive (sticky) ends:

• Single-stranded overhangs

• Can base-pair with complementary DNA

Overhang:

• Extra single-stranded DNA after staggered cut

Blunt ends:

• No overhang

• Straight cut across both strands

What are factors that impact efficiency of digestion?

• Temperature → optimal usually 37°C

• Buffer → provides correct conditions

• pH (~8) → required for enzyme activity

• Salt concentration (Na⁺/K⁺) → affects enzyme specificity

• Mg²⁺ ions → required cofactor

• DNA concentration → too much DNA reduces efficiency

• Enzyme amount → must be sufficient

• Glycerol concentration → too high (>5%) inhibits enzyme

• Incubation time → usually ~1 hour

How do you determine whether your RE digestion was successful?

Run samples on agarose gel electrophoresis

Successful digestion:

• DNA appears as distinct bands

• Band sizes match expected fragments

How do you determine whether RE digestion was complete or incomplete?

Complete digestion:

• Only expected fragment bands appear

Incomplete digestion:

• Mixture of:

  • Undigested plasmid (supercoiled band)

  • Partially digested fragments

 How did you set up your specific digests?

Setup:

• Each reaction contained:

  • DNA (~500 ng)

  • Buffer (2.5 µL)

  • Restriction enzyme(s)

  • Water to 25 µL total

• Tubes included:

  • Single digests (BamHI or HindIII)

  • Double digest (BamHI + HindIII)

  • Incubated at 37°C for 1 hour

What was the need to do both single and double digests?

Why single vs double digests:

• Single digest:

  • Determines number of cut sites for each enzyme

• Double digest:

  • Determines relative positions of sites

  • Helps build a restriction map

What was the exact aim of restriction enzyme digestion?

Aim of experiment:

• To characterize plasmid DNA

• Identify structure by restriction mapping

• Confirm identity of plasmid (pAMP or pKAN)

How have REs revolutionized the field of gene cloning?

• Allow DNA to be cut at precise locations

• Enable insertion of genes into plasmids

• Make recombinant DNA technology possible

• Allow:

  • Gene cloning

  • DNA mapping

  • Genetic engineering

•  Foundation of modern molecular biology