Team:Newcastle/PCR
From 2010.igem.org
(Difference between revisions)
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==GoTaq PCR== | ==GoTaq PCR== | ||
- | ===Materials to add accordingly | + | ===Materials to add accordingly=== |
# 37.5 µl of distilled H2O | # 37.5 µl of distilled H2O | ||
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# 1 µl template DNA | # 1 µl template DNA | ||
- | ===Conditions for ThermoCycler | + | ===Conditions for ThermoCycler=== |
# Initialise - 95°C for 2 minutes. | # Initialise - 95°C for 2 minutes. | ||
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==Phusion PCR== | ==Phusion PCR== | ||
- | ===Materials to add accordingly | + | ===Materials to add accordingly=== |
# 27.5 µl of distilled H2O | # 27.5 µl of distilled H2O | ||
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# 5 µl backward primer | # 5 µl backward primer | ||
# 1 µl template DNA | # 1 µl template DNA | ||
- | #0.5 µl of Fusion | + | # 0.5 µl of Fusion |
- | ===Conditions for ThermoCycler | + | ===Conditions for ThermoCycler=== |
# Initialise - 98°C for 30 seconds. | # Initialise - 98°C for 30 seconds. |
Revision as of 14:04, 27 July 2010
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Contents |
GoTaq PCR
Materials to add accordingly
- 37.5 µl of distilled H2O
- 10 µl of 5x GoTaq Buffer
- 1 µl of nucleotide DNTP
- 2.5 µl forward primer
- 2.5 µl backward primer
- 1 µl template DNA
Conditions for ThermoCycler
- Initialise - 95°C for 2 minutes.
- Denature - 95°C for 30 seconds.
- Anneal - 52°C for 30 seconds (melting temperature, Tm, of template)
- Extension - 75°C for 30 seconds
- Extension finish - 75°C for 5 minutes
- Hold - 4°C
Steps 2 to 4 are repeated for 30 cycles before continuing to step 5.
Phusion PCR
Materials to add accordingly
- 27.5 µl of distilled H2O
- 10 µl of 5x Buffer
- 1 µl of nucleotide DNTP
- 5 µl forward primer
- 5 µl backward primer
- 1 µl template DNA
- 0.5 µl of Fusion
Conditions for ThermoCycler
- Initialise - 98°C for 30 seconds.
- Denature - 98°C for 10 seconds.
- Anneal - x°C for 20 seconds (melting temperature, Tm, of template)
- Extension - 72°C for 30 seconds per kb
- Extension finish - 72°C for 5-10 minutes
- Hold - 4°C
Steps 2 to 4 are repeated for 30 cycles before continuing to step 5.