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From 2014.igem.org

BYU 2014 Notebook EDIT Procedures

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Q5 PCR 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

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50 uL Reaction

uL Q5 Polymerase (add right before you start the PCR reaction. Keep on ice until you are ready to do so.)

For 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 (this is called a “hot start”). Abundant templates only require 20-25 cycles for amplification; dilute/complex templates require 35-40 cycles. Extension times vary depending on target size.