Team:BYU Provo/Notebook/CommonProcedures

From 2014.igem.org

(Difference between revisions)
Line 116: Line 116:
<li>1 ul appropriate diluted template DNA</li>
<li>1 ul appropriate diluted template DNA</li>
<li>0.5 ul Phusion Polymerase</li></ul>
<li>0.5 ul Phusion Polymerase</li></ul>
 +
<ul><li><b>Standard Phusion 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>
 +
 +
 +
</html>
</html>

Revision as of 01:16, 22 July 2014


BYU 2014 Notebook

EDIT Procedures

Home Team Official Team Profile Project Parts Modeling Notebook Safety Attributions

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

Retrieved from "http://2014.igem.org/Team:BYU_Provo/Notebook/CommonProcedures"