Fong:PCR: Difference between revisions

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=Deng's PCR Protocol=


==Preparation of DNA==
 
==Preparation==
*Prepare DNA from cell sample during mid-log phase, or as extraction kit designates.
*Prepare DNA from cell sample during mid-log phase, or as extraction kit designates.
*Use Spectrophotometer to determine concentration and total amount of extracted DNA.
*Use Spectrophotometer to determine concentration and total amount of extracted DNA.


==Program Thermocycler==
==Program Thermocycler==
*Use standard program.
*Use standard program.
*Use T<sub>annealing</sub> that is 5°C lower than the least T<sub>m</sub> of the primers.
*Use T<sub>annealing</sub> that is 5°C lower than the least T<sub>m</sub> of the primers.
*Use final extension time of 1:00.
*Use final extension time of 1 min.
*May use extended primary melting time, to ensure full denaturation of template.
*May use extended primary melting time, to ensure full denaturation of template.
*'''Use at least 1 min extension cycle per kb of product.'''
==PCR Taq Mix==
==PCR Taq Mix==


{| cellpadding="2" align="right" border="0.9" padding="3"
{| cellpadding="2" align="right" border="0.9" padding="3"
|+ PCR Reaction Mix Ingredients
|+ Taq Mix
|-
|-
! Name !! Volume
! Name !! Volume
Line 24: Line 27:
| 0.5 μL
| 0.5 μL
|-
|-
! F-primer
! F-primer (25nM)
| 1.25 μL
| 1.25 μL
|-
|-
! R-primer
! R-primer (25nM)
| 1.25 μL
| 1.25 μL
|-
|-
Line 45: Line 48:
| 25 μL
| 25 μL
|}
|}


#Add all reagents to labeled PCR reaction tube, adding Taq polymerase last.
#Add all reagents to labeled PCR reaction tube, adding Taq polymerase last.
Line 55: Line 60:
*Be sure to run at least four varying Mg<sup>2+</sup> concentrations in order to optimize yield and fidelity.
*Be sure to run at least four varying Mg<sup>2+</sup> concentrations in order to optimize yield and fidelity.
*Run a gel using some product and product digestion to verify fragment size and proper restriction sites.
*Run a gel using some product and product digestion to verify fragment size and proper restriction sites.
==PCR Phusion High-Fidelity Polymerase Mix==
{| cellpadding="2" align="right" border="0.9" padding="3"
|+ Phusion Mix
|-
! Name !! Volume
|-
! Phusion buffer
| 5 μL
|-
! dNTP
| 0.2 μL
|-
! F-primer (25nM)
| 0.5 μL
|-
! R-primer (25nM)
| 0.5 μL
|-
! Phusion Polymerase
| 0.25 μL
|-
! DNA
| 1 μL
|-
! DMSO
| 0.75 μL
|-
! Mg<sup>2+</sup>
| 0 || 0.5 || 1.0 μL
|-
! H<sub>2</sub>O
| 16.8 || 16.3 || 15.8 μL
|-
! Total V
| 25 μL
|}
#Add all reagents to labeled PCR reaction tube, adding Phusion polymerase last.
#Place in the thermocycler and select cycle, making any changes as noted above.
#Run PCR reaction.
#Enjoy your brand-new DNA!
#????
#PROFT
*Be sure to run varying Mg<sup>2+</sup> concentrations in order to optimize yield and fidelity.
*Run a gel using some product and template to verify fragment size and proper restriction sites.


==References==
==References==

Revision as of 08:22, 15 July 2009

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Preparation

  • Prepare DNA from cell sample during mid-log phase, or as extraction kit designates.
  • Use Spectrophotometer to determine concentration and total amount of extracted DNA.


Program Thermocycler

  • Use standard program.
  • Use Tannealing that is 5°C lower than the least Tm of the primers.
  • Use final extension time of 1 min.
  • May use extended primary melting time, to ensure full denaturation of template.
  • Use at least 1 min extension cycle per kb of product.

PCR Taq Mix

Taq Mix
Name Volume
Taq buffer 2.5 μL
dNTP 0.5 μL
F-primer (25nM) 1.25 μL
R-primer (25nM) 1.25 μL
Mg2+ 0 0.5 1.0 1.5
Taq 0.2 0.2 0.2 0.2
H2O 17.3 16.8 16.3 15.8
DNA 2 2 2 2
Total V 25 μL


  1. Add all reagents to labeled PCR reaction tube, adding Taq polymerase last.
  2. Place in the thermocycler and select cycle, making any changes as noted above.
  3. Run PCR reaction.
  4. Enjoy your brand-new DNA!
  5. ????
  6. PROFT
  • Be sure to run at least four varying Mg2+ concentrations in order to optimize yield and fidelity.
  • Run a gel using some product and product digestion to verify fragment size and proper restriction sites.

PCR Phusion High-Fidelity Polymerase Mix

Phusion Mix
Name Volume
Phusion buffer 5 μL
dNTP 0.2 μL
F-primer (25nM) 0.5 μL
R-primer (25nM) 0.5 μL
Phusion Polymerase 0.25 μL
DNA 1 μL
DMSO 0.75 μL
Mg2+ 0 0.5 1.0 μL
H2O 16.8 16.3 15.8 μL
Total V 25 μL



  1. Add all reagents to labeled PCR reaction tube, adding Phusion polymerase last.
  2. Place in the thermocycler and select cycle, making any changes as noted above.
  3. Run PCR reaction.
  4. Enjoy your brand-new DNA!
  5. ????
  6. PROFT


  • Be sure to run varying Mg2+ concentrations in order to optimize yield and fidelity.
  • Run a gel using some product and template to verify fragment size and proper restriction sites.


References

Contact

Yu Deng

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