Difference between revisions of "Team:Hong Kong UCCKE/Protocols"
| Line 33: | Line 33: | ||
</div> | </div> | ||
<div class="collapse-card"> | <div class="collapse-card"> | ||
| − | <div class="title"> | + | <div class="title">Gel Electrophoresis</div> |
<div class="body">Suspendisse rhoncus massa augue, eget efficitur nisi pellentesque a. Nam euismod purus eu tortor imperdiet finibus. Sed efficitur a tortor at mollis. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Fusce a aliquam ante, ac suscipit risus. Aenean venenatis porta arcu id iaculis. Phasellus tempus scelerisque nisl a malesuada. Vestibulum auctor scelerisque tortor eu faucibus. Quisque porttitor arcu sed nulla pharetra, sit amet sollicitudin quam suscipit. Praesent malesuada auctor metus dictum interdum. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec ac lectus quis elit consectetur tempor. Maecenas ac massa id tortor feugiat auctor.</div> | <div class="body">Suspendisse rhoncus massa augue, eget efficitur nisi pellentesque a. Nam euismod purus eu tortor imperdiet finibus. Sed efficitur a tortor at mollis. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Fusce a aliquam ante, ac suscipit risus. Aenean venenatis porta arcu id iaculis. Phasellus tempus scelerisque nisl a malesuada. Vestibulum auctor scelerisque tortor eu faucibus. Quisque porttitor arcu sed nulla pharetra, sit amet sollicitudin quam suscipit. Praesent malesuada auctor metus dictum interdum. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec ac lectus quis elit consectetur tempor. Maecenas ac massa id tortor feugiat auctor.</div> | ||
</div> | </div> | ||
<div class="collapse-card"> | <div class="collapse-card"> | ||
| − | <div class="title"> | + | <div class="title">Gel purification</div> |
<div class="body">Suspendisse rhoncus massa augue, eget efficitur nisi pellentesque a. Nam euismod purus eu tortor imperdiet finibus. Sed efficitur a tortor at mollis. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Fusce a aliquam ante, ac suscipit risus. Aenean venenatis porta arcu id iaculis. Phasellus tempus scelerisque nisl a malesuada. Vestibulum auctor scelerisque tortor eu faucibus. Quisque porttitor arcu sed nulla pharetra, sit amet sollicitudin quam suscipit. Praesent malesuada auctor metus dictum interdum. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec ac lectus quis elit consectetur tempor. Maecenas ac massa id tortor feugiat auctor.</div> | <div class="body">Suspendisse rhoncus massa augue, eget efficitur nisi pellentesque a. Nam euismod purus eu tortor imperdiet finibus. Sed efficitur a tortor at mollis. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Fusce a aliquam ante, ac suscipit risus. Aenean venenatis porta arcu id iaculis. Phasellus tempus scelerisque nisl a malesuada. Vestibulum auctor scelerisque tortor eu faucibus. Quisque porttitor arcu sed nulla pharetra, sit amet sollicitudin quam suscipit. Praesent malesuada auctor metus dictum interdum. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec ac lectus quis elit consectetur tempor. Maecenas ac massa id tortor feugiat auctor.</div> | ||
</div> | </div> | ||
<div class="collapse-card"> | <div class="collapse-card"> | ||
| − | <div class="title"> | + | <div class="title">Ligation</div> |
| − | <div class="body"> | + | <div class="body"><b>Materials needed</b><br> |
| + | <ul><li>T4 DNA Ligase Reaction Buffer</li> | ||
| + | <li>T4 DNA Ligase</li> | ||
| + | <li>Microcentrifuge</li> | ||
| + | <li>Thermocycler, or waterbath and thermometer</li> | ||
| + | <li>Restriction Digest: pSB1C3 linearized plasmid backbone</li> | ||
| + | <li>Restriction Digest: Insert</li></ul><br> | ||
| + | <b>Protocol</b><br> | ||
| + | <ol start="1"><li>Add 2ul from the pSB1C3 linearized plasmid backbone digest.</li> | ||
| + | <li>Add 3.3ul from the Part digest.</li> | ||
| + | <li>Add 1ul of T4 DNA Ligase Reaction Buffer.</li> | ||
| + | <li>Add 0.5ul of T4 DNA Ligase (keep this at -20°C until use!).</li> | ||
| + | <li>Mix by gently pipetting up and down 3x. Do not vortex; this inactivates the enzymes. Place tube in microcentrifuge for a quick 5 second spin or flick the tube to collect the mixture at the bottom.</li> | ||
| + | <li> Incubate at 16°C for 30 minutes, then at 80°C for 20 minutes using a thermocycler.</li></ol> | ||
| + | </div> | ||
</div> | </div> | ||
<div class="collapse-card"> | <div class="collapse-card"> | ||
Revision as of 08:48, 14 October 2017
Restriction Enzyme Digestion
Materials needed
Protocols
- 70% ethanol
- Paper towels
- Ice
- Container for ice
- Lab marker/Sharpie
- pSB1C3 linearized plasmid backbone (25ng/ul) (kit)
- Part (25ng/ul)
- 0.6ml tubes (kit)
- 10X Red Buffer
- NEB enzymes: EcoRI, PstI
- Thermocycler, or waterbath and thermometer
Protocols
- Clean the lab bench by wiping down with 70% ethanol and paper towels.
- Keep all enzymes and buffers used in this section on ice.
- Prepare a reaction mix by adding reagents in the order indicated in Table 1.
- Incubate at 37°C for 20 minutes.
Gel Electrophoresis
Suspendisse rhoncus massa augue, eget efficitur nisi pellentesque a. Nam euismod purus eu tortor imperdiet finibus. Sed efficitur a tortor at mollis. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Fusce a aliquam ante, ac suscipit risus. Aenean venenatis porta arcu id iaculis. Phasellus tempus scelerisque nisl a malesuada. Vestibulum auctor scelerisque tortor eu faucibus. Quisque porttitor arcu sed nulla pharetra, sit amet sollicitudin quam suscipit. Praesent malesuada auctor metus dictum interdum. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec ac lectus quis elit consectetur tempor. Maecenas ac massa id tortor feugiat auctor.
Gel purification
Suspendisse rhoncus massa augue, eget efficitur nisi pellentesque a. Nam euismod purus eu tortor imperdiet finibus. Sed efficitur a tortor at mollis. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Fusce a aliquam ante, ac suscipit risus. Aenean venenatis porta arcu id iaculis. Phasellus tempus scelerisque nisl a malesuada. Vestibulum auctor scelerisque tortor eu faucibus. Quisque porttitor arcu sed nulla pharetra, sit amet sollicitudin quam suscipit. Praesent malesuada auctor metus dictum interdum. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec ac lectus quis elit consectetur tempor. Maecenas ac massa id tortor feugiat auctor.
Ligation
Materials needed
Protocol
- T4 DNA Ligase Reaction Buffer
- T4 DNA Ligase
- Microcentrifuge
- Thermocycler, or waterbath and thermometer
- Restriction Digest: pSB1C3 linearized plasmid backbone
- Restriction Digest: Insert
Protocol
- Add 2ul from the pSB1C3 linearized plasmid backbone digest.
- Add 3.3ul from the Part digest.
- Add 1ul of T4 DNA Ligase Reaction Buffer.
- Add 0.5ul of T4 DNA Ligase (keep this at -20°C until use!).
- Mix by gently pipetting up and down 3x. Do not vortex; this inactivates the enzymes. Place tube in microcentrifuge for a quick 5 second spin or flick the tube to collect the mixture at the bottom.
- Incubate at 16°C for 30 minutes, then at 80°C for 20 minutes using a thermocycler.
Your title goes here
Suspendisse rhoncus massa augue, eget efficitur nisi pellentesque a. Nam euismod purus eu tortor imperdiet finibus. Sed efficitur a tortor at mollis. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Fusce a aliquam ante, ac suscipit risus. Aenean venenatis porta arcu id iaculis. Phasellus tempus scelerisque nisl a malesuada. Vestibulum auctor scelerisque tortor eu faucibus. Quisque porttitor arcu sed nulla pharetra, sit amet sollicitudin quam suscipit. Praesent malesuada auctor metus dictum interdum. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec ac lectus quis elit consectetur tempor. Maecenas ac massa id tortor feugiat auctor.
Your title goes here
Suspendisse rhoncus massa augue, eget efficitur nisi pellentesque a. Nam euismod purus eu tortor imperdiet finibus. Sed efficitur a tortor at mollis. Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Fusce a aliquam ante, ac suscipit risus. Aenean venenatis porta arcu id iaculis. Phasellus tempus scelerisque nisl a malesuada. Vestibulum auctor scelerisque tortor eu faucibus. Quisque porttitor arcu sed nulla pharetra, sit amet sollicitudin quam suscipit. Praesent malesuada auctor metus dictum interdum. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Donec ac lectus quis elit consectetur tempor. Maecenas ac massa id tortor feugiat auctor.
