Team:BYU Provo/Notebook/CommonProcedures
From 2014.igem.org
(Difference between revisions)
Line 88: | Line 88: | ||
<li>Mix well then add 2.5 ul REDTAQ polymerase</li></ul> | <li>Mix well then add 2.5 ul REDTAQ polymerase</li></ul> | ||
<p>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.</p> | <p>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.</p> | ||
+ | <ul><li><b>Standard PCR program:</li></b></ul> | ||
+ | <ol><li>95°C for 2 min</li> | ||
+ | <li>95°C for 30 sec</li> | ||
+ | <li>55°C for 30 sec</li> | ||
+ | <li>72°C for 2 min</li> | ||
+ | <li>Repeat (2-4) 35 times</li> | ||
+ | <li>72°C for 5 min</li> | ||
+ | <li>4°C for forever</li></ol> | ||
<h2>Q5 PCR Reaction (50 uL Reaction)</h2> | <h2>Q5 PCR Reaction (50 uL Reaction)</h2> |
Revision as of 01:06, 22 July 2014
BYU 2014 Notebook |
||||||||||||
| ||||||||||||
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:
- 95°C for 2 min
- 95°C for 30 sec
- 55°C for 30 sec
- 72°C for 2 min
- Repeat (2-4) 35 times
- 72°C for 5 min
- 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