CHAPTER 2 (PCR INGREDIENTS)

Question 1

one of the ingredients in PCR is buffer and MgCl2. what does the MgCl2 do

Answer 1

1. the Mg2+ in MgCl2 acts as a cofactor for Taq polymerase. its required for Taq to function.
2. the Mg2+ influence the binding efficiency and stability of the primer-template complex.
3. Mg2+ helps stabilise interactions between primer and DNA template

Question 2

what happened if MgCl is high or low

Answer 2

high: more product, less specific
low: less product, more specific

Question 3

how much dNTPs should be used?

Answer 3

50 to 500mM each

50mM is enough to make 6 microgram of product

Question 4

what does Taq do with dNTPs?

Answer 4

Taq adds dNTPs one by one to extend DNA strand

Question 5

what does it mean by Taq polymerase is heat stable?

Answer 5

it withstand high temp needed for DNA denaturation without getting denatured itself

Question 6

whats the function of Taq polymerase

Answer 6

synthesize new DNA strands by adding nucleotides to primers / catalyse a reaction by adding nucle from free 3’OH end of primer

Question 7

Taq polymerase works at __C

Answer 7

72 C

Question 8

whats primers

Answer 8

short, single-stranded sequences of nucleotides (usually around 18-30 bases long) that are complementary to a specific region of the DNA template. They serve as the starting point for DNA synthesis during processes like PCR

Question 9

does Taq polymerase has proof-reading ability. why?

Answer 9

naur. bcs of its enzyme behaviour

Question 10

Taq polymerase naturally adds

Answer 10

a single adenine at the 3’ end (A overhangs)

Question 11

what does the extra A at the end can be used for?

Answer 11

in T/A cloning systems

Question 12

what do we design the primers for?

Answer 12

to anneal to specific sequences.

Question 13

pcr is specific bcs of

Answer 13

the primers

Question 14

only the seq between two primers are

Answer 14

amplified

Question 15

what are the guidelines of primers design?

Answer 15

1. check orientation of primers
2. 20-30bp long
3. 40-50% GC content
4. avoid internal structure
5. avoid complementary between primers
6. avoid extensive GC at 3’ end

Question 16

whats melting temp and how to calculate it

Answer 16

temperature at which
half of the DNA duplex (primer + template)
dissociates into single strands.

Melting temperature (Tm) = 4 (G+C) + 2 (A+T)

Question 17

formula of annealing temp

Answer 17

Ta= Tm-5

Question 18

whats hairpin loop?

Answer 18

If there are complementary sequences within a primer, it will make internal structure called hairpin loop

Question 19

whats primer dimer

Answer 19

If there are complimentary sequences in two primers used (one primer for each DNA strand), the primers will hybridize with each other thus forming primer-dimmers and will not be available for binding with template