AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol
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| - | http://stanxterm.aecom.yu.edu/wiki/data/Product_manuals_attach/pfuturbo.pdf PCR manual | + | [http://stanxterm.aecom.yu.edu/wiki/data/Product_manuals_attach/pfuturbo.pdf PCR manual] |
==Materials== | ==Materials== | ||
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| align="center" style="background:#f0f0f0;"|'''Component Amount per reaction''' | | align="center" style="background:#f0f0f0;"|'''Component Amount per reaction''' | ||
|- | |- | ||
| - | | Distilled water (dH2O) 40.6 μl | + | | Distilled water (dH2O) 40.6 μl |
|- | |- | ||
| - | | 10× cloned Pfu reaction buffer 5.0 μl | + | | 10× cloned Pfu reaction buffer 5.0 μl |
|- | |- | ||
| - | | dNTPs (25 mM each dNTP) 0.4 μl | + | | dNTPs (25 mM each dNTP) 0.4 μl |
|- | |- | ||
| - | | DNA template (100 ng/μl) 1.0 μl | + | | DNA template (100 ng/μl) 1.0 μl |
|- | |- | ||
| - | | Primer #1 (100 ng/μl) 1.0 μl | + | | Primer #1 (100 ng/μl) 1.0 μl |
|- | |- | ||
| - | | Primer #2 (100 ng/μl) 1.0 μl | + | | Primer #2 (100 ng/μl) 1.0 μl |
|- | |- | ||
| PfuTurbo® DNA polymerase (2.5 U/μl) 1.0 μl (2.5 U) | | PfuTurbo® DNA polymerase (2.5 U/μl) 1.0 μl (2.5 U) | ||
|- | |- | ||
| - | | Total reaction volume 50 μl | + | | Total reaction volume 50 μl |
|} | |} | ||
Revision as of 13:25, 15 October 2012
Contents |
Materials
- primers
- plasmid
- dexynucleoside triphosphate mixture (dNTPs)
Equipment
PCR tubes temperature cycler
Parameters
- for DNA vectors (plasmids) of less than 10 kbases)
- The primers must each be at 100-200 ng/μL
- 100-250 μM of each dNTPs
- denaturing temperature is 95 °C
- extension temperature is 72 °C (extension time 1 min per kb)
- 25-30 cycles
- enzyme concentration is 2.5 units of enzyme/ 50 μL of reaction for vector targets up to 19 kb and must be the last component added to PCR mixture (ie after dNTPS) to prevent degradation of the primers.
Protocol
- prepare the reaction mixture of 50 μL
- add the components in order
Component Amount per reaction
| Component Amount per reaction |
| Distilled water (dH2O) 40.6 μl |
| 10× cloned Pfu reaction buffer 5.0 μl |
| dNTPs (25 mM each dNTP) 0.4 μl |
| DNA template (100 ng/μl) 1.0 μl |
| Primer #1 (100 ng/μl) 1.0 μl |
| Primer #2 (100 ng/μl) 1.0 μl |
| PfuTurbo® DNA polymerase (2.5 U/μl) 1.0 μl (2.5 U) |
| Total reaction volume 50 μl |
- immediately before thermal cycling, aliquot 50 μl of the reaction mixture into a sterile 0.5 mL micro centrifuge tube
- thermocycle
- for targets less than 10 kb vector DNA
1. 2 min at 95 °C 2. repeat this 30 times
a) 30 s at 95 °C b) 30 s at Tm - 5 °C c) 1 min at 72 oC for each 1 kb
3. 10 min at 72 °C 4. set temperature to 0 °C
Notes
- Backbone size w/o insert (bp):7328 bp for ADA
