Team:TU Darmstadt/Protocols/PCR

(Difference between revisions)

Contents 1 Polymerase Chain Reaction (PCR) 1.1 Working principle 1.2 Mixtures 1.2.1 PCR I NEB Phusion (50 µL batch) 1.2.2 PCR II House-Taq (50 µL batch) 1.2.3 PCR III NEB Q5 (50 µL batch) 1.2.4 SOE PCR 1.3 PCR Programs 1.3.1 Standard program 1.3.2 Colony PCR 1.3.3 SOE PCR

Polymerase Chain Reaction (PCR)

Polymeriase chain reaction the most common method for DNA amplification. Detailed information is available at [http://en.wikipedia.org/wiki/Polymerase_chain_reaction wikipedia].

Working principle

The PCR is typically based on four steps:

1. heating to 95°C to separate the DNA double strands
2. annealing at a temperature specific for the primers to anneal specifically to the template DNA (e.g. 63°C); too low or too high temperature does increases unspecific annealing or prevents any annealing
3. amplification at the optimal working temperature of the polymerase (Taq, Phusion eg.)(72-74°C)
4. a number of repeat from 1-3 for 25-35 times

Mixtures

PCR I NEB Phusion (50 µL batch)

• • dNTPs 1 µL
  • Primer up (10 picomol/µL) 1 µL
  • Primer lo (10 picomol/µL) 1 µL
  • Template 1 µL
  • MgCl₂(50mM) 0.5 µL
  • DMSO 1.5 µL
  • 5x Phusion Buffer / GC Buffer 10µL
  • ddH₂O 34.5 µL
  • NEB Phusion Polymerase 0.5 µL

PCR II House-Taq (50 µL batch)

• • dNTPs 1µL
  • Primer up (10 picomol/µL) 1 µL
  • Primer lo (10 picomol/µL) 1 µL
  • Template 1 µL
  • DMSO 1.5 µL
  • 5x GoTaq Buffer 10 µL
  • ddH₂O 35.5 µL
  • House-Taq Polymerase 1 µL

PCR III NEB Q5 (50 µL batch)

• • dNTPs 1 µL
  • Primer up (10 picomol/µL) 1 µL
  • Primer lo (10 picomol/µL) 1 µL
  • Template 1 µL
  • 5x Q5 Reaction Buffer 10 µL
  • Q5 GC Enhancer 10µL
  • ddH₂O 34.5 µL
  • NEB Q5 Polymerase 0.5 µL

SOE PCR

Unlike PCR I-III the premix is done without adding any primers, but just the two annealing templates. After 10 cycles the Primer up and lo are added.

PCR Programs

Standard program

1. 98°C, 30s
2. Denature: 98°C, 10s
3. Annealing: T_A, t_A
4. Elongation: 72°C, t_E

   step 2 to 4 is repeated 30-35 times

5. 72°C, 5min
6. 10°C continuously

Colony PCR

1. 98°C, 5min
2. Denature: 98°C, 10s
3. Annealing: T_A, t_A
4. Elongation: 72°C, t_E

step 2 to 4 is repeated 30-35 times

1. 72°C, 5min
2. 10°C continuously

SOE PCR

1. 98°C, 30s
2. Denature: 98°C, 10s
3. Annealing: T_A1, t_A1
4. Elongation: 72°C, t_E1

   step 2 to 4 is repeated 10 times then the primers up and lo are added

5. Denature: 98°C, 10s
6. Annealing: T_A2, t_A2
7. Elongation: 72°C, t_E2

   step 5 to 7 is repeated 30-35 times

8. 72°C, 5min
9. 10°C continuously

Annotations: T_A - Annealing temperature; t_A - Annealing time;t_E - Elongation time; T_A, t_A=f(Primer up and Primer lo); t_E=f(Polymerase, gene length)