Difference between revisions of "Skatebro:Ligation"

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(Tips)
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[[Skatebro:Project08]]
 
  
==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
 
 
==Controls==
 
*You'll know if it worked after transforming :)
 
 
==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
 

Latest revision as of 02:08, 9 February 2008