Fong:PCR: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
|||
Line 1: | Line 1: | ||
{{Template:Fong}} | {{Template:Fong}} | ||
==Preparation | |||
==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 | *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" | ||
|+ | |+ 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
<owwmenu font="arial, helvetica, sans-serif" bold="1"
color="White" bgcolor="gray" hovercolor=#FF9933 bghovercolor="black" topfontsize="7" fontSize="7" pagewidth="750" image="Cth.JPG" lab="Fong"> Home= Lab Members=People Research=Research Internal=Internal, Protocols=Protocols, Chemical Inventory=Inventory Links=Links
</owwmenu>
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
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 |
- Add all reagents to labeled PCR reaction tube, adding Taq 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 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
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 |
- 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 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.