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From 2014.igem.org

BYU 2014 Notebook EDIT Procedures

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TAQ PCR (50 uL Reaction)

• 35 ul ddH20
• 10 ul 5X REDTAQ buffer (mix well before use!!)
• 1.0 ul 10 mM dNTP’s
• 1 ul each Primer (50 uM stock)
• 1 ul appropriate diluted template DNA
• Mix well then add 2.5 ul REDTAQ polymerase

Set up reactions on ice, and keep them on ice until placing them on the PCR machine which has been pre-warmed to 94°C. Abundant templates only require 20-25 cycles for amplification; dilute/complex templates require 35-40 cycles. Extension times vary depending on target size.

• Standard PCR program:

1. 95°C for 2 min
2. 95°C for 30 sec
3. 55°C for 30 sec
4. 72°C for 2 min
5. Repeat (2-4) 35 times
6. 72°C for 5 min
7. 4°C for forever

Q5 PCR Reaction (50 uL Reaction)

• 10 uL 5x Q5 Reaction Buffer
• 1 uL dNTPs
• 1 uL Forward Primer
• 1 uL Reverse Primer
• 10 uL Q5 Enhancer
• 23.5 uL ddH2O
• 1-2 uL Template
• Add ?uL Q5 Polymerase right before you start the PCR reaction.

Set up reactions on ice, making sure to keep Q5 Polymerase on ice until needed.

Phusion PCR

• 35 ul ddH20
• 10 ul 5X Phusion GC buffer
• 1.0 uL DMSO
• 1.5 ul 10 mM dNTP’s
• 1 ul each Primer (50 uM stock)
• 1 ul appropriate diluted template DNA
• 0.5 ul Phusion Polymerase

• Standard Phusion PCR program:

1. 95°C for 2 min
2. 95°C for 30 sec
3. 55°C for 30 sec
4. 72°C for 2 min
5. Repeat (2-4) 35 times
6. 72°C for 5 min
7. 4°C for forever