Wittrup: PCR: Difference between revisions
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'''Materials''' | '''Materials''' | ||
Taq polymerase (usually 5 U per | |||
Taq polymerase (usually 5 U per 0.001 mL) and 10X buffer | |||
MgCl2 | MgCl2 | ||
forward primer | forward primer | ||
reverse primer | reverse primer | ||
DNA template | DNA template | ||
PCR grade water | PCR grade water | ||
dNTP mix or each of dATP, dCTP, dGTP, dTTP | dNTP mix or each of dATP, dCTP, dGTP, dTTP | ||
2.5 M betaine (optional) | 2.5 M betaine (optional) | ||
'''Mix the components of each | DMSO (optional) | ||
'''Mix the components of each 0.050 mL reaction on ice:''' | |||
The final concentration of each of the reagents are as follows: | |||
10X buffer---------------1X | |||
10X buffer | |||
MgCl2 | MgCl2--------------------final concentration 1.5 to 2.5 mM | ||
forward primer | |||
reverse primer | forward primer----------0.5 uM | ||
dNTP | |||
DNA template | reverse primer----------0.5 uM | ||
water | |||
Taq Polymerase 2.5 to 5 Unit | dNTP---------------------10 mM each of dATP, dGTP, dTTP, dCTP | ||
DNA template------------typically 10 ng | |||
water----------------------add water to 0.049 mL volume | |||
Taq Polymerase---------2.5 to 5 Unit (usually 0.001 mL) | |||
The use of DMSO and betaine usually improve specificity of the annealing reaction. If the use of DMSO and betaine are desired, the final concentration of DMSO and betaine should be 3% (v/v) and 1 M. | |||
Proceed with thermal cycling conditions outlined below: | Proceed with thermal cycling conditions outlined below: | ||
Denature 1 min at 94 | |||
Anneal | Denature-------1 min at 94 degree Celsius | ||
Extend | |||
Anneal----------1 min at the proper annealing temperature (depend on the primers) | |||
Extend----------1 min for each 1 kb at 72 degree Celsius | |||
Repeat for 30 cycles (typical amplification) | Repeat for 30 cycles (typical amplification) | ||
Final extension for 5 to 10 min at 72 | |||
Final extension for 5 to 10 min at 72 degree Celsius. |
Latest revision as of 13:41, 24 September 2006
Materials
Taq polymerase (usually 5 U per 0.001 mL) and 10X buffer
MgCl2
forward primer
reverse primer
DNA template
PCR grade water
dNTP mix or each of dATP, dCTP, dGTP, dTTP
2.5 M betaine (optional)
DMSO (optional)
Mix the components of each 0.050 mL reaction on ice:
The final concentration of each of the reagents are as follows:
10X buffer---------------1X
MgCl2--------------------final concentration 1.5 to 2.5 mM
forward primer----------0.5 uM
reverse primer----------0.5 uM
dNTP---------------------10 mM each of dATP, dGTP, dTTP, dCTP
DNA template------------typically 10 ng
water----------------------add water to 0.049 mL volume
Taq Polymerase---------2.5 to 5 Unit (usually 0.001 mL)
The use of DMSO and betaine usually improve specificity of the annealing reaction. If the use of DMSO and betaine are desired, the final concentration of DMSO and betaine should be 3% (v/v) and 1 M.
Proceed with thermal cycling conditions outlined below:
Denature-------1 min at 94 degree Celsius
Anneal----------1 min at the proper annealing temperature (depend on the primers)
Extend----------1 min for each 1 kb at 72 degree Celsius
Repeat for 30 cycles (typical amplification)
Final extension for 5 to 10 min at 72 degree Celsius.