MOL BIO LAB L7 (Midterms- PCR)

Question 1

A fundamental technique in molecular biology and genetics.

Answer 1

PCR

Question 2

A widely used molecular biology technique for amplifying DNA. The procedure involves a series of temperature cycles, typically consisting of denaturation, annealing, and extension steps.

Answer 2

PCR

Question 3

The initial step of Denaturation involves heating the reaction mixture around _____ to denature the DNA

Answer 3

94- 98C

Question 4

The temperature is lowered to around ______ to aIIow the primers to anneal (bind) to their complementary sequences on the single-stranded DNA template.

Answer 4

50- 65°C

Question 4

These steps (denaturation, annealing, and extension) are typically repeated for ______ to achieve a significant amplification of the target DNA.

Answer 4

20-40 cycles

Question 5

To setup a PCR reaction, a _______ is prepared.

Answer 5

PCR master mix

Question 5

In extension, the temperature is raised to_______, and DNA polymerase extends the primers by adding nucleotides, thus synthesizing new DNA strands.

Answer 5

72-75°C

Question 6

Enumerate the reagents included in the master mix

Answer 6

DNA template, primers, DNA polymerase, nucleotides, and buffer.

Question 7

The precise composition of the master mix depends on the?

Answer 7

specific PCR protocol and reagents used.

Question 8

These are needed to be calculated to optimize the PCR reaction

Answer 8

• Concentration of primers
• DNA template
• dNTPs (deoxynucleotide triphosphate)
• Buffer components

Question 9

The total volume of the PCR reaction depends on the type of PCR machine used but is typically?

Answer 9

20-50 uL

Question 9

The master mix is prepared in a _______ to avoid contamination. It’s crucial to keep reagents on _____ and to use dedicated pipettes and tips to prevent cross-contamination.

Answer 9

sterile environment; ice

Question 9

Primers should not form _________ or ___________

Answer 9

secondary structures; self-complementarity

Question 10

Primers are typically _______ nucleotides in length.

Answer 10

18-24

Question 11

The GC content should be around _____ to optimize primer annealing.

Answer 11

40-60%

Question 12

The melting temperature of primers should be similar,
typically within?

Answer 12

2°C of each other

Question 13

Primers should be designed to specifically anneal to the _________, avoiding non-specific binding.

Answer 13

target DNA

Question 14

Adding _______ region at the ________ can enhance primer stability

Answer 14

GC-rich region; 3’ end

Question 15

During the PCR reaction, what factors are needed to be considered?

Answer 15

• Template DNA
• Cycling Conditions
• Positive and Negative Controls
• Reaction Volume
• Thermal Cyclers
• Data Analysis

Question 16

TRUE OR FALSE: Use a reliable thermal cycler that can accurately control weather and time machine

Answer 16

FALSE. It should be can accurately control temperature and time cycles

Question 17

After PCR, the amplified products can be analyzed by _______, real-time PCR, or ________to verify the presence of the target
DNA.

Answer 17

gel electrophoresis; sequencing

Question 18

NOTE: Keep in mind that PCR protocols and reagents may evolve, so it’s important to consult the latest literature and manufacturer’s instructions for the most up-to-date information and best practices

Question 19

This is the type of DNA preferred as the template DNA

Answer 19

Purified DNA with minimal contaminants

Question 20

These characteristics of template DNA are crucial (2pts)

Answer 20

Quality and Quantity

Question 21

Include _________and ________in your PCR to ensure the reaction is working and to check for contamination.

Answer 21

positive; negative controls

Question 22

Ensure that the _______ is consistent and that you are using appropriate reaction tubes or plates.

Answer 22

reaction volume

Question 23

Series of Temperature cycles (3 pts)

Answer 23

• Denaturation
• Annealing
• Extension

Question 24

Considerations in creating an effective primer design

Answer 24

• Primer length
• GC content
• Tm (Melting temp)
• Avoiding Self-Complementarity
• Specificity
• GC clamp