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Team:TU Darmstadt/Protocols/PCR
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# Annealing: T<sub>A</sub>, t<sub>A</sub> | # Annealing: T<sub>A</sub>, t<sub>A</sub> | ||
# Elongation: 72°C, t<sub>E</sub> | # Elongation: 72°C, t<sub>E</sub> | ||
| - | + | :step 2 to 4 is repeated 30-35 times | |
# 72°C, 5min | # 72°C, 5min | ||
# 10°C continuously | # 10°C continuously | ||
| Line 98: | Line 95: | ||
# Annealing: T<sub>A</sub>, t<sub>A</sub> | # Annealing: T<sub>A</sub>, t<sub>A</sub> | ||
# Elongation: 72°C, t<sub>E</sub> | # Elongation: 72°C, t<sub>E</sub> | ||
| - | : step 2 to 4 is repeated 30-35 times | + | :step 2 to 4 is repeated 30-35 times |
# 72°C, 5min | # 72°C, 5min | ||
# 10°C continuously | # 10°C continuously | ||
| Line 107: | Line 104: | ||
# Annealing: T<sub>A1</sub>, t<sub>A1</sub> | # Annealing: T<sub>A1</sub>, t<sub>A1</sub> | ||
# Elongation: 72°C, t<sub>E1</sub> | # Elongation: 72°C, t<sub>E1</sub> | ||
| - | + | : step 2 to 4 is repeated 10 times then the primers up and lo are added | |
# Denature: 98°C, 10s | # Denature: 98°C, 10s | ||
# Annealing: T<sub>A2</sub>, t<sub>A2</sub> | # Annealing: T<sub>A2</sub>, t<sub>A2</sub> | ||
# Elongation: 72°C, t<sub>E2</sub> | # Elongation: 72°C, t<sub>E2</sub> | ||
| - | + | :step 5 to 7 is repeated 30-35 times | |
# 72°C, 5min | # 72°C, 5min | ||
# 10°C continuously | # 10°C continuously | ||
'''Annotations:''' T<sub>A</sub> - Annealing temperature; t<sub>A</sub> - Annealing time;t<sub>E</sub> - Elongation time; T<sub>A</sub>, t<sub>A</sub>=f(Primer up and Primer lo); t<sub>E</sub>=f(Polymerase, gene length) | '''Annotations:''' T<sub>A</sub> - Annealing temperature; t<sub>A</sub> - Annealing time;t<sub>E</sub> - Elongation time; T<sub>A</sub>, t<sub>A</sub>=f(Primer up and Primer lo); t<sub>E</sub>=f(Polymerase, gene length) | ||
Latest revision as of 21:29, 26 September 2012
Contents |
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:
- heating to 95°C to separate the DNA double strands
- 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
- amplification at the optimal working temperature of the polymerase (Taq, Phusion eg.)(72-74°C)
- 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
- 98°C, 30s
- Denature: 98°C, 10s
- Annealing: TA, tA
- Elongation: 72°C, tE
- step 2 to 4 is repeated 30-35 times
- 72°C, 5min
- 10°C continuously
Colony PCR
- 98°C, 5min
- Denature: 98°C, 10s
- Annealing: TA, tA
- Elongation: 72°C, tE
- step 2 to 4 is repeated 30-35 times
- 72°C, 5min
- 10°C continuously
SOE PCR
- 98°C, 30s
- Denature: 98°C, 10s
- Annealing: TA1, tA1
- Elongation: 72°C, tE1
- step 2 to 4 is repeated 10 times then the primers up and lo are added
- Denature: 98°C, 10s
- Annealing: TA2, tA2
- Elongation: 72°C, tE2
- step 5 to 7 is repeated 30-35 times
- 72°C, 5min
- 10°C continuously
Annotations: TA - Annealing temperature; tA - Annealing time;tE - Elongation time; TA, tA=f(Primer up and Primer lo); tE=f(Polymerase, gene length)
