Recombinant DNA Techniques: PCR

What ions are required for PCR?

Mg

What is the purpose of Mg ions in PCR?

How would you describe Taq and Pfu DNA Polymerases?

Thermostable

Where do the primers bind?

To both the 5’ and the 3’ end of the melted strands

What is a dNTP

deoxynucleotide triphosphate (free nucleotide)

Which thermostable DNA Polymerase is proofreading?

Pfu

Which thermostable DNA Polymerase isn’t proofreading?

Taq

How many cycles are peformed in PCR

30

What are the three cycles of PCR?

denaturation, annealing and extension

What temperature does denaturation occur?

over 90

What temperature does annealing occur

50

What temperature does extension occur?

72

How long is a primer typically and what GC content does it have?

17-30bp, at least 40% GC content

Why is being 17-30bp and over 40% GC content important for a primer?

prevents primers from binding to themselves (primer dimers) and the formation of hairpin loops (too short for self binding)

creates solid annealing

How is a primer Tm calculated?

What end of the DNA strand do the primers bind to?

3’ end

What does it mean for a primer in PCR to be either forward or reverse?

Why is PCR not perfectly exponential growth?

renaturation of DNA can occur preventing replication

Why must the Tm of the two primers be within 2 degrees of each other

What happens if the primer Tm is exceeded

What happens if the PCR temperature is too far under the primer Tm

What is the pGEM-T System

A commersaily avalible open vector which is complementayr to the addittional nucleotide added by Taq

in the FASTA format, which strand is the coding sequence?

The top strand

What is a 5’ 3’ exonuclease activity?

proofreading

What secondary activity does Taq Polymerase have

a terminal transferase added on the 3’ end

(additional nucleotide requiring no template)

What is the prefered addittional nucleotide to be added by Taq and why?

A, prevent self bindign

What is the name of the additional nucleotide added by Taq

terminal transferase

What are common problems with PCR

digestion efficiency, ligation efficiency, avoiding recirculation of open vector

How do you avoid recirclisation of a cut vector?

Use two restriction enzymes with non-compatible ends

What does a phosphatase do?

How does a phosphatase prevent a vector from recirculating

What do you need to ensure is removed before electroporation

What is electroporation?

What is chemical transformation

How is chemical transformation acheived

What does the calculation for transformation efficency measure?

CFU/micro gram of DNA

What is the coefficient for pico

What does bacterial competency refer to?

Ability to transform

What is a transformant?