Skatebro:Ligation: Difference between revisions
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*Keep buffer/enzyme on ice block for the entire process | *Keep buffer/enzyme on ice block for the entire process | ||
*Do calculations based on fresh dna spec reading | *Do calculations based on fresh dna spec reading | ||
*50ng of backbone and insert (but extra insert can't hurt) | *~50ng of backbone and insert (but extra insert can't hurt) | ||
*10μL total volume | *10μL total volume | ||
Revision as of 20:50, 31 January 2008
Tips
- Keep buffer/enzyme on ice block for the entire process
- Do calculations based on fresh dna spec reading
- ~50ng of backbone and insert (but extra insert can't hurt)
- 10μL total volume
Steps
- Add in order: (10μL little pcr-sized tube)
- ?? μL water to bring the total volume to 10μL
- ?? μL cut, cleaned-up insert (whatever necessary to get 50ng or slightly >50 ng)
- ?? μL cut, cleaned-up backbone (whatever necessary to get 50ng)
- 1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells like wet dog)
- .5μL T4 DNA ligase enzyme
- Leave tubes at room temperature for about 15 minutes
