Skatebro:Ligation

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(Tips)
Current revision (06:08, 9 February 2008) (view source)
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[[Skatebro:Project08]]
 
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==Tips==
 
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*Keep buffer/enzyme on ice block for the entire process
 
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*Do calculations based on fresh dna spec reading
 
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*~50ng of backbone and insert (but extra insert can't hurt)
 
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*10μL total volume
 
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==Controls==
 
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*You'll know if it worked after transforming :)
 
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==Steps==
 
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#Add in order: (10μL little pcr-sized tube)
 
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#*?? μL water to bring the total volume to 10μL
 
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#*?? μL cut, cleaned-up insert (whatever necessary to get 50ng or slightly >50 ng)
 
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#*?? μL cut, cleaned-up backbone (whatever necessary to get 50ng)
 
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#*1μL T4 DNA ligase buffer (vortex well before adding, make sure it smells like wet dog)
 
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#*.5μL T4 DNA ligase enzyme
 
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#Leave tubes at room temperature for about 15 minutes
 

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