"
Team:Newcastle/PCR
From 2010.igem.org
(Difference between revisions)
| Line 1: | Line 1: | ||
{{Team:Newcastle/mainbanner}} | {{Team:Newcastle/mainbanner}} | ||
| - | + | =GoTaq PCR= | |
| - | + | ==Materials required== | |
| + | Add the following according: | ||
# 37.5 µl of distilled H<sub>2</sub>O | # 37.5 µl of distilled H<sub>2</sub>O | ||
| Line 12: | Line 13: | ||
# 1 µl template DNA | # 1 µl template DNA | ||
| - | + | ==Conditions for ThermoCycler== | |
# Initialise - 95°C for 2 minutes. | # Initialise - 95°C for 2 minutes. | ||
| Line 23: | Line 24: | ||
Steps 2 to 4 are repeated for 30 cycles before continuing to step 5. | Steps 2 to 4 are repeated for 30 cycles before continuing to step 5. | ||
| - | + | =Phusion PCR= | |
| - | + | ==Materials required== | |
| + | Add the following according: | ||
# 27.5 µl of distilled H<sub>2</sub>O | # 27.5 µl of distilled H<sub>2</sub>O | ||
| Line 35: | Line 37: | ||
# 0.5 µl of Fusion | # 0.5 µl of Fusion | ||
| - | + | ==Conditions for ThermoCycler== | |
# Initialise - 98°C for 30 seconds. | # Initialise - 98°C for 30 seconds. | ||
Revision as of 14:51, 28 July 2010
| |||||||||||||
| |||||||||||||
Contents |
GoTaq PCR
Materials required
Add the following according:
- 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 required
Add the following according:
- 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.
   
|
    
|
