View source for Restriction Enzyme Based Cloning

#If insert is PCR, run PCR then PCR purify (Qiagen kit).  Elute in 30 uL EB
#Digest both vector (~1ug) and insert in appropriate buffer 1-2h at 37C.  Add 1 uL of CIAP to the vector for the last ~10min at 37C.  
#Gel purify both vector and insert (Qiagen kit).  Elute in 30 uL
#Combine 3-6 uL insert with 1-2 uL vector.  You want about a 3x excess of insert to vector.  Place at 50-65C for 5-10 min to help sticky end binding. Also do a no insert negative control. 
#Add 2 uL ligase buffer (single use aliquots) and water/EB to 10 uL final volume.  Add 1 uL T4 DNA Ligase (Invitrogen).  
#Incubate 1h-O/N at 16C (water bath in cold room).  
#Transform 5-10 uL into 50 uL DH5a cells (subcloning efficiency):
##Make 50 uL aliquots
##Add DNA and mix gently
##Incubate on ice for 30min
##Heat shock for 20s at 37C
##Place on ice for 2min
##Add 450 uL LB
##Incubate at 37C for 1h with shaking
##Plate 500 uL and grow O/N at 37C on appropriate plates 
#Pick several colonies and digest to verify insert.  Sequence if necessary.