PCR MASTERMIX PREPARATION AND TEMPLATE ADDITION

Question 1

batch mixture of PCR reagents at optimal
concentrations that can be prepared
and divided among many PCR tubes
or 96-well PCR plates

Answer 1

PCR master mix, sometimes known
as super mix or ready mix

Question 2

he master mix
usually includes

Answer 2

DNA polymerase,
dNTPs, MgCl2 and buffer

Question 3

Maintains the optimal pH level and provides the necessary ions (such as potassium and
chloride) to stabilize the DNA polymerase and facilitate its enzymatic activity throughout the
PCR reaction

Answer 3

Buffer

Question 4

helps maintain the stability of the reaction components and supports efficient DNA
amplification

Answer 4

Buffer

Question 5

helps maintain the stability of the reaction components and supports efficient DNA
amplification

Answer 5

Buffer

Question 6

Magnesium ions (Mg2+) are cofactors required for the activity of most DNA polymerases

Answer 6

Magnesium ions (Mg2+)

Question 7

is added to the PCR mix to provide the optimal concentration of magnesium ions,
which is critical for the efficiency and specificity of DNA synthesis.

Answer 7

MgCl2

Question 8

During PCR, dNTPs are incorporated by the DNA polymerase into the newly synthesized DNA
strands, complementing the template DNA sequence

Answer 8

Nucleotides (dNTPs)

Question 9

are short, single-stranded DNA sequences that bind to complementary sequences
on the target DNA.

Answer 9

Primers

Question 10

In PCR, _____are designed to flank the region of interest,
defining the boundaries of the DNA segment to be amplified.

Answer 10

In PCR, two primers (forward and reverse) are designed to flank the region of interest,
defining the boundaries of the DNA segment to be amplified.

Question 11

They provide the starting point for DNA synthesis by the DNA polymerase.

Answer 11

Primers

Question 12

short, single-stranded DNA molecules labeled with a fluorescent dye and a quencher
molecule.

Answer 12

short, single-stranded DNA molecules labeled with a fluorescent dye and a quencher
molecule.

Question 13

They hybridize specifically to the target DNA sequence during PCR and emit fluorescence
upon binding, allowing for real-time detection of DNA amplification.

Answer 13

Probes

Question 14

These can include PCR enhancers to improve amplification efficiency or overcome PCR
inhibitors, stabilizers to maintain the integrity of the reaction components during storage, or
detergents to aid in cell lysis and DNA release.

Answer 14

Optional additives

Question 15

This enzyme is essential for synthesizing new DNA strands by adding nucleotides to the growing
DNA chain

Answer 15

DNA polymerase

Question 16

Additives included to improve the stability of the enzyme during storage and handling.

Answer 16

Additives included to improve the stability of the enzyme during storage and handling.

Question 17

Serves as a reference to confirm that the PCR reaction is working correctly and that the assay
conditions are suitable for amplification.

Answer 17

. POSITIVE CONTROL
COMPONENTS
Serves as a reference

Question 18

contains in vitro transcriptional RNA containing target genes or cloned plasmids containing genome
sequences and internal standard gene fragments

Answer 18

3. POSITIVE CONTROl

Question 19

helps to detect any contamination or non-specific amplification that may occur in the absence of the
target sequence.

Answer 19

NEGATIVE CONTRO

Question 20

It serves as a reference to ensure that any observed PCR products in the experimental samples

Answer 20

NEGATIVE CONTROL

Question 21

refers to the process of introducing the target DNA or RNA template into a
polymerase chain reaction (PCR) mastermix.

Answer 21

PCR template addition

Question 22

he template DNA or RNA contains the region of interest that will be amplified during the PCR reaction.