Berkeley LBL/PCRphusion
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< Berkeley LBL(Difference between revisions)
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*5x GC Buffer may be used in replacement of HF Buffer for high GC content for Phusion kit | *5x GC Buffer may be used in replacement of HF Buffer for high GC content for Phusion kit | ||
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2. ''Run PCR machine'' | 2. ''Run PCR machine'' | ||
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*Immediately place on ice or in 4 ºC after removing from PCR machine | *Immediately place on ice or in 4 ºC after removing from PCR machine | ||
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'''*Cool Start Method for PCR (for more accurate amplification):''' | '''*Cool Start Method for PCR (for more accurate amplification):''' | ||
Latest revision as of 21:02, 17 October 2007
PCR (Phusion Polymerase)
1. Set Up PCR Reaction:
1 uL template DNA
10 uL Phusion 5x HF Buffer
1 uL 10mM dNTPs
5 uL Primer Mix
0.5 uL DMSO (optional)
0.5 uL Phusion Polymerase
Add H2O to total volume 50 uL
- Add the polymerase last and carefully.
- DMSO is used only for high GC content
- 5x GC Buffer may be used in replacement of HF Buffer for high GC content for Phusion kit
2. Run PCR machine
- Use 30 sec/kb for extension
- General cycle:
1. Initial Denature 98ºC 30 sec
2. Denaturation 98 ºC 8 sec
3. Annealing Annealing Temp 30 sec
4. Extension 72 ºC 30sec/kb
5. Repeat steps 2-4 for an additional 29 cycles
6. Final Extension 72 ºC 10 min
7. 4 ºC forever
- Immediately place on ice or in 4 ºC after removing from PCR machine
*Cool Start Method for PCR (for more accurate amplification):
1. Keep all reagents on ice until use
2. Prepare the reaction mixture on ice
3. Set PCR machine ready to start with the designated program
4. Place tubes in PCR machine and start cycle immediately
