AU Biomaterials Design Lab:Protocols/PCR Mutation Protocol
From OpenWetWare
< AU Biomaterials Design Lab:Protocols(Difference between revisions)
Current revision (13:29, 15 October 2012) (view source) (→Protocol) |
|||
| 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}} | ||
| Line 45: | Line 45: | ||
|} | |} | ||
| - | + | *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 | ||
Current revision
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
