Restriction Enzyme Based Cloning: Difference between revisions

Line 1:

#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

#Gel purify both vector and insert (Qiagen kit).

#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~~.

:*Run out on gel (see [[Preparing an Agarose Gel]]

#Add 2 uL ligase buffer (single use aliquots) ~~and water/EB to 10 uL final volume~~. Add 1 uL T4 DNA Ligase (Invitrogen).

:*On a gel box cut out appropriate bands and place in clean eppendorf tubes. Try to limit the exposure to UV light

#Incubate 1h-O/N at 16C (water bath in cold room).

:*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.

#Transform 5-10 uL into 50 uL ~~DH5a~~ cells ~~(subcloning efficiency)~~:

:*Place at 55-65C until gel is dissolved (5-10 min)

:*Add to pink purification column, wash and elute in 30 uL

#Combine 6 uL insert with 1 uL vector. Place at 50-65C for 5-10 min to help sticky end binding.

#Add 2 uL ligase buffer (single use aliquots).

#Add 1 uL T4 DNA Ligase (Invitrogen).

#Incubate 2h-O/N at 16C (water bath in cold room).

#Transform 5-10 uL into 50 uL supercompetent cells:

##Make 50 uL aliquots

##Add DNA and mix gently

##Incubate on ice for 30min

##Heat shock for ~~20s~~ at ~~37C~~

##Heat shock for 45s at 42C

##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

##Plate 500 uL and grow O/N at 37C on appropriate antibiotic plates

#Pick several colonies and digest to verify insert. Sequence if necessary.

@@ Line 1: / Line 1: @@
 #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
+#Gel purify both vector and insert (Qiagen kit).
-#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.
+:*Run out on gel (see [[Preparing an Agarose Gel]]
-#Add 2 uL ligase buffer (single use aliquots) and water/EB to 10 uL final volume.  Add 1 uL T4 DNA Ligase (Invitrogen).
+:*On a gel box cut out appropriate bands and place in clean eppendorf tubes.  Try to limit the exposure to UV light
-#Incubate 1h-O/N at 16C (water bath in cold room).
+:*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.
-#Transform 5-10 uL into 50 uL DH5a cells (subcloning efficiency):
+:*Place at 55-65C until gel is dissolved (5-10 min)
+:*Add to pink purification column, wash and elute in 30 uL
+#Combine 6 uL insert with 1 uL vector.  Place at 50-65C for 5-10 min to help sticky end binding.
+#Add 2 uL ligase buffer (single use aliquots).
+#Add 1 uL T4 DNA Ligase (Invitrogen).
+#Incubate 2h-O/N at 16C (water bath in cold room).
+#Transform 5-10 uL into 50 uL supercompetent cells:
 ##Make 50 uL aliquots
 ##Add DNA and mix gently
 ##Incubate on ice for 30min
-##Heat shock for 20s at 37C
+##Heat shock for 45s at 42C
 ##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
+##Plate 500 uL and grow O/N at 37C on appropriate antibiotic plates
 #Pick several colonies and digest to verify insert.  Sequence if necessary.