PCR

Question 1

What are the reagents for PCR

Answer 1

Pair of primers (1 forward, 1 reverse)
DNA template (doesn’t have to be pure)
Polymerase
Reaction buffer

Question 2

How do you chose the primer pairs

Answer 2

18-30 based
40-60% GC but no long 3 end GC clamp (avoids primer dimers)
TM should be around 55°C and should be similar for both forward and reverse
Avoid hairpins (middle section having complementary bases)

Question 3

What is TM calculation

Answer 3

Can be done through in-silico PCR but general formula:

TM = 4(G + C) + 2(A + T)

Question 4

What are the different types of thermostable polymerases

Answer 4

Pfu turbo-
High accuracy
Used to amplify complex genomic targets up to 10kb or vector targets up to 15kb

Pfu-
High accuracy and lowest error rate
Used for PCR blunt end cloning

TaqPlus-
Very accurate and very fast

Taq-
Very standard not very accurate

Question 5

What are the different amplifications in PCR

Answer 5

Specific amplification-
Specific primers are used to bind to target DNA so it can allow for extension along template through polymerase to create a new DNA strand indentical to target

Exponential amplification-
Repeated cycling of denaturing, annealing and extension each cycle roughly doubling number of DNA

Question 6

How does thermal cycler work

Answer 6

Has slots to insert PCR mixture
Starts at 4 degrees
Increases to 95 to denature
Goes down to 55 for annealing
Goes up to 72 for extension
Then repeats