Plasmid Analysis Lab

Question 1

What are the four main steps in the lab protocol?

Answer 1

Transform 🟢 → Grow 🟡 → Purify 🔵 → Digest & Electrophoresis 🔴

Question 2

What does the mnemonic ‘This Industry Hates Integrity’ represent?

Answer 2

Thaw ❄️ - Incubate 🧬 - Heat Shock 🔥 - Incubate 🌡️

Question 3

What is the first step in the transformation of plasmid DNA into E. coli?

Answer 3

Thaw E. coli cells on ice (10 min)

Question 4

What is the purpose of heat shock during transformation?

Answer 4

Creates temporary pores in the cell membrane

Question 5

What does the mnemonic ‘Pardon My Gigantic Cock’ represent?

Answer 5

Pick 📍 - Mix 🧪 - Grow Colonies 🧫

Question 6

What is the purpose of incubating transformed bacteria in outgrowth medium?

Answer 6

Allows bacteria to recover and express antibiotic resistance genes

Question 7

What is the first step in plasmid purification (Miniprep)?

Answer 7

Centrifuge bacterial culture (5 min)

Question 8

What does the mnemonic ‘Let’s Nuke White People’ represent?

Answer 8

Lysis 🟥 - Neutralization 🟦 - Wash 🪣 - Purification 🔵

Question 9

What is the role of NaOH in the lysis buffer (A2)?

Answer 9

Denatures chromosomal & plasmid DNA

Question 10

What does the mnemonic ‘Pigs Run Every Institution’ represent?

Answer 10

Prepare 🧪 - Restrict ✂️ - Electrophorese ⚡ - Interpret 🔎

Question 11

What is the purpose of restriction enzyme digestion?

Answer 11

Cuts plasmid DNA at specific sites to confirm presence of an insert

Question 12

What happens to DNA during gel electrophoresis?

Answer 12

DNA moves towards positive electrode due to its negative charge

Question 13

What is a plasmid?

Answer 13

A small, circular DNA molecule used in genetic engineering

Question 14

What is a selectable marker?

Answer 14

A gene that allows only transformed bacteria to survive on selective media

Question 15

What are restriction enzymes?

Answer 15

Proteins that cut DNA at specific sequences

Question 16

What is a competent cell?

Answer 16

Specially prepared E. coli that can take up DNA

Question 17

Fill in the blank: The heat shock method creates temporary pores in the bacterial membrane at ______°C.

Answer 17

42

Question 18

What is the purpose of the washing step in plasmid purification?

Answer 18

Removes contaminants using AW & A4 buffers

Question 19

What is the expected outcome of gel electrophoresis?

Answer 19

Separation of DNA fragments based on size

Question 20

What can cause no colonies on the transformation plate?

Answer 20

No plasmid DNA was added, heat shock ineffective, or wrong antibiotic used

Question 21

What is the significance of using a silica membrane in plasmid purification?

Answer 21

DNA binds to silica in high salt conditions while impurities are washed away

Question 22

What does genomic DNA contamination during purification indicate?

Answer 22

Vortexing after adding A2 or incomplete neutralization step

Question 23

What are common issues that can arise during gel electrophoresis?

Answer 23

• No bands visible
• Smearing in gel
• Faint bands

Question 24

What is the role of the loading dye in gel electrophoresis?

Answer 24

To visualize the DNA and ensure proper loading amount

Question 25

Why do supercoiled plasmids run faster than linear DNA in a gel?

Answer 25

Supercoiled DNA is more compact

Question 26

Fill in the blank: The main purpose of the outgrowth phase after transformation is to express ______.

Answer 26

antibiotic resistance genes

Question 27

What is the expected size of DNA fragments compared to a molecular weight marker?

Answer 27

To verify successful plasmid digestion

Question 28

What can cause unexpected bands in a restriction digest?

Answer 28

* Partial digestion * Plasmid supercoiling * Star activity

Question 29

What is the purpose of including an undigested plasmid control in gel electrophoresis?

Answer 29

To confirm that restriction digestion occurred correctly

Question 30

What is the consequence of incomplete lysis during plasmid prep?

Answer 30

Low DNA yield and contamination with genomic DNA

Question 31

What happens if we forget to add a molecular weight marker in gel electrophoresis?

Answer 31

We cannot determine the size of our DNA fragments