AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol: Difference between revisions
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(New page: ==Materials== * primers * plasmid *dexynucleoside triphosphate mixture (dNTPs) ==Equipment== PCR tubes temperature cycler ==Directions== *for DNA vectors (plasmids) of less than 10 kba...) |
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http://stanxterm.aecom.yu.edu/wiki/data/Product_manuals_attach/pfuturbo.pdf PCR manual | |||
==Materials== | ==Materials== | ||
* primers | * primers | ||
* plasmid | * plasmid | ||
| Line 14: | Line 15: | ||
**The primers must each be at 100-200 nh/mL | **The primers must each be at 100-200 nh/mL | ||
** dNTPS 100-250 μM of each dNTPs | ** dNTPS 100-250 μM of each dNTPs | ||
** denaturing temperature is 95 | ** denaturing temperature is 95 °C | ||
** extension temperature is 72 oC | ** extension temperature is 72 °C (extension time 1 min per kb) | ||
** 25-30 cycles | |||
** enzyme concentration is 2.5 units of enxyme/ 50 μL of reaction for vector targets up to 19 kb and must be hte last component added to PCR mixture (ie after dNTPS) | |||
==Protocol== | |||
# prepare the reaction mixture of 50 μL | |||
##add the components in order | |||
Component Amount per reaction | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
Revision as of 10:22, 15 October 2012
http://stanxterm.aecom.yu.edu/wiki/data/Product_manuals_attach/pfuturbo.pdf PCR manual
Materials
- primers
- plasmid
- dexynucleoside triphosphate mixture (dNTPs)
Equipment
PCR tubes temperature cycler
Directions
- for DNA vectors (plasmids) of less than 10 kbases)
- The primers must each be at 100-200 nh/mL
- dNTPS 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 enxyme/ 50 μL of reaction for vector targets up to 19 kb and must be hte last component added to PCR mixture (ie after dNTPS)
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
