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copied over protocol
#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.
#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.