AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol: Difference between revisions
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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== | ||
| Line 22: | Line 22: | ||
==Protocol== | ==Protocol== | ||
*prepare the reaction mixture of 50 μL | |||
**add the components in order | |||
Component Amount per reaction | Component Amount per reaction | ||
{| {{table}} | {| {{table}} | ||
| 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 | ||
|} | |} | ||
*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 <br> | |||
1. 2 min at 95 °C | 2. repeat this 30 times<br> | ||
2. repeat this 30 times | a) 30 s at 95 °C <br> | ||
a) 30 s at 95 °C | b) 30 s at Tm - 5 °C <br> | ||
b) 30 s at Tm - 5 °C | c) 1 min at 72 oC for each 1 kb<br> | ||
c) 1 min at 72 oC for each 1 kb | 3. 10 min at 72 °C<br> | ||
3. 10 min at 72 °C | 4. set temperature to 0 °C<br> | ||
4. set temperature to 0 °C | |||
==Notes== | ==Notes== | ||
* Backbone size w/o insert (bp):7328 bp for ADA | * Backbone size w/o insert (bp):7328 bp for ADA | ||
Latest revision as of 10:29, 15 October 2012
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
