915
edits
Changes
updated with gel purification
#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 :*Run out on gel (see [[Preparing an Agarose Gel]]:*On a gel box cut out appropriate bands and place in clean eppendorf tubes. Try to limit the exposure to UV light:*Weigh out gel piece. For each mg add 3 uL of QG (orange) buffer. For example, for a 0.1g piece add 300 uL buffer.:*Place at 55-65C until gel is dissolved (5-10 min):*Add to pink purification column, wash and 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 1h2h-O/N at 16C (water bath in cold room). #Transform 5-10 uL into 50 uL DH5a supercompetent cells (subcloning efficiency):
##Make 50 uL aliquots
##Add DNA and mix gently
##Incubate on ice for 30min
##Heat shock for 20s 45s at 37C42C
##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 antibiotic plates
#Pick several colonies and digest to verify insert. Sequence if necessary.