PCR

Question 1

enumerate the steps of PCR

Answer 1

1. denaturation
2. annealing
3. extension

Question 2

this step involves heating the reaction mixture to — the DNA, separating the double-stranded into single strand. what is the temp for this step?

Answer 2

DENATURATION
94-98C

Question 3

the temperature is lowered around — to allow primers to anneal (bind) to their complementary sequences on the single stranded DNA template.

Answer 3

50 - 65 C

Question 4

the temperature is raised to —, and the DNA polymerase extends the primers by adding nucleotides, thus — new DNA strands

Answer 4

72-75 C
synthesizing new DNA strands

Question 5

the steps of PCR are repeated for — to achieve a significant amplification of the target DNA

Answer 5

20-40 cycles

Question 6

maxter mix iincludes all the reagents required for PCR such as — enumerate!

Answer 6

• DNA template
• primers
• DNA polymerase
• nucleotides
• buffer

Question 7

enumerate the key condiserations in preparation of PCR master mix

Answer 7

• reagent concentrations
• total reaction volume
• mix preparation

Question 8

total volume of PCR is typically —

Answer 8

20-50 uL

Question 9

enumerate the principles in primer design (6)

Answer 9

• primer length
• GC content
• melting temp
• avoiding self complementarity
• specificity
• GC clamp

Question 10

primer length

Answer 10

18-24 nucleotides

Question 11

GC content

Answer 11

40-60%

Question 12

melting temperature

Answer 12

2C each other

Question 13

adding a GC-rich region at the 3’ end can enhance?

Answer 13

primer stability

Question 14

factors needed to be considered in PCR reaction (6)

Answer 14

• template DNA
• cycling conditions
• positive and negative controls
• reaction volume
• thermal cyclers
• data analysis

Question 15

preferred template DNA

Answer 15

purified DNA with minimal contaminants

Question 16

purpose of positive and negative control

Answer 16

1. to check contamination
2. ensure the reaction is working

Question 17

after PCR, the amplified products can be analyzed by?

Answer 17

1. gel electrophoresis
2. real-time PCR
3. sequencing

to verify the presence of the target DNA

Question 18

materials needed for PCR

Answer 18

• PCR reagents (DNA pol, dNTPs, buffer)
• microcentrifuge tube
• electrophoresis equipment
• agarose gel and buffer
• DNA ladder marker
• Gel electrophoresis staining solution
• pipettes and tips
• safety equipments
• DNA template
• forward and reverse primers

Question 19

portion of DNA to make several copies

Answer 19

amplification