PCR and Gel Electrophoresis

Question 1

What is the purpose of PCR?

Answer 1

To amplify a single copy of DNA, allowing for analysis

Question 2

What is required for PCR?

Answer 2

• dNTP (nucleotides for extension)
• buffer
• Taq Polymerase
• Primers (both forward and reverse)
• DNA to be amplified
• Thermocycler (or similar)

Question 3

Describe the process of PCR (lol)

Answer 3

1. Denaturation
   High temperatures denature DNA by degrading hydrogen bonds between the strands, separating them
2. Annealing
   Primers (short, complementary sequences) bind to flanking regions upon the single strands of DNA. DNA polymerase can only add to nucleotides to double stranded DNA.
   Forward primers bind to 5’ end, reverse primers bind to 3’ end

3. Extension 
Taq polymerase (a thermostable DNA polymerase) extends DNA sequences in a 5' --> 3' direction, using dNTPs.

Question 4

What is the gel composed of for gel electrophoresis?

Answer 4

TAE Gel (TRIS, Acetic acid, edta) 
TRIS = buffer 
acetic acid (technically forms Tris-acetate) = buffer that prevents hydrolysis 
EDTA = chelates cations (e.g. Mg2+ and Ca2+), these are required for DNAses to work; sequestration prevents DNAses from working and therefore protects DNA from damage

Question 5

What is the purpose of Sybr-safe in gel-electrophoresis?

Answer 5

Sybr-safe is a DNA-binding dye that will bind to DNA and fluoresce when viewed under UV light.
Allows bands to be visualised (representing DNA).

Question 6

What is the purpose of marker?

Answer 6

Marker, also referred to as DNA ladder, contain fragments of known length, allowing for estimation of molecular weights