Difference between revisions of "Team:Ionis Paris/Protocol 10"
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<li> | <li> | ||
| − | <p>25 cycles of :<br/> - 98°C 10s<br/> - Provided Ta temperature 10-30s<br/> - 72°C; 20/ | + | <p>25 cycles of :<br/> - 98°C 10s<br/> - Provided Ta temperature 10-30s<br/> - 72°C; 20/30s per kB (amplification)</p> |
</li> | </li> | ||
<li> | <li> | ||
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</ul> | </ul> | ||
| + | </aside> | ||
</div> | </div> | ||
<h4 class="sidebar_Hd">Sources</h4> | <h4 class="sidebar_Hd">Sources</h4> | ||
| − | <p><li><a href="https://www.neb.com/applications/cloning-and-synthetic-biology/site-directed-mutagenesis">NEB Site directed Mutagenesis overview</a></li></p> | + | <p><li><a href="https://www.neb.com/applications/cloning-and-synthetic-biology/site-directed-mutagenesis"><font color="DeepPink">NEB Site directed Mutagenesis overview</font></a></li></p> |
| − | <p><li><a href="https://www.neb.com/protocols/2013/01/26/q5-site-directed-mutagenesis-kit-quick-protocol-e0554">NEB Site directed Mutagenesis quick protocol</a></li></p> | + | <p><li><a href="https://www.neb.com/protocols/2013/01/26/q5-site-directed-mutagenesis-kit-quick-protocol-e0554"><font color="DeepPink">NEB Site directed Mutagenesis quick protocol</font></a></li></p> |
| − | <p><li><a href="http://nebasechanger.neb.com/">NEBaseChanger</a></li></p> | + | <p><li><a href="http://nebasechanger.neb.com/"><font color="DeepPink">NEBaseChanger</font></a></li></p> |
| − | + | ||
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| − | <h4>IONIS | + | <h4>iGEM IONIS</h4> |
| − | <p> We're a group of six different schools from the IONIS Education Group. For this competition we wanted to take advantages of the multiple schools and activity field given by the IONIS education group to create a solid project.</p> | + | <p> We're a group of six different schools from the IONIS Education Group. For this |
| − | + | competition we wanted to take advantages of the multiple schools and activity field | |
| + | given by the IONIS education group to create a solid project.</p> | ||
| + | <a href="https://2016.igem.org/Team:Ionis_Paris/Team">Read More</a> | ||
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| − | <li><a href="https://2016.igem.org/Team:Ionis_Paris/ | + | <li><a href="https://2016.igem.org/Team:Ionis_Paris/HPintro">Human |
| − | <li><a href="https://2016.igem.org/Team | + | Practice</a></li> |
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Latest revision as of 19:53, 19 October 2016
Primers should be designed with 5´ ends annealing back-to-back. It is recommend to use the NEB online design software, NEBaseChanger™. Prepare the following reaction mix: Perform a thermocycling as follows: Initial denaturation: 98°C for 30s 25 cycles of : 72°C for 2 min Hold: 4°C Prepare the following reaction mix: Incubate for 5 min at room temperature Add 5μL of KLD mix to 50μL of chemically-competent cells.
Protocol 10: Site directed Mutagenesis
Aim: Modify a given section of a plasmid
Exponential amplification
Component
Volume
Final concentration
Q5 Hot Start High-Fidelity 2X Master Mix
12.5 µL
1X
10 μM Forward Primer
1.25 μL
0.5 μM
10 μM Reverse Primer
1.25 μL
0.5 μM
Template DNA (1–25 ng/μl)
1 μL
1-25 ng
Nuclease-free water
9 µL
- 98°C 10s
- Provided Ta temperature 10-30s
- 72°C; 20/30s per kB (amplification) KLD Reaction
Component
Volume
Final concentration
PCR product
1 µL
2X KLD Reaction Buffer
5 µL
1X
10X KLD Enzyme Mix
1 µL
1X
Nuclease-free Water
3 µL
Transformation
Incubate on ice for 30 minutes.
Heat shock at 42°C for 30 seconds.
Incubate on ice for 5 minutes.
Add 950μL SOC, gently shake at 37°C for 1 hour.
Spread 40–100 μL onto appropriate selection plate, incubate overnight at 37°C.
