Skatebro:Ligation: Difference between revisions
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(New page: Skatebro:Project08 ===Tips=== *Do calculations based on dna spec reading *Want 50 nanograms of backbone and insert *Want 10 microL total volume) |
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==Tips== | |||
*Do calculations based on dna spec reading | *Keep buffer/enzyme on ice block entire time | ||
* | *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) | |||
#*Enough water to bring the total volume to 10μL | |||
#*50ng (or >50 ng) of cut, cleaned-up insert | |||
#*50ng of cut, cleaned-up backbone | |||
#*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 | |||
Revision as of 20:42, 31 January 2008
Tips
- Keep buffer/enzyme on ice block entire time
- 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)
- Enough water to bring the total volume to 10μL
- 50ng (or >50 ng) of cut, cleaned-up insert
- 50ng of cut, cleaned-up backbone
- 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
