In-Fusion Cloning – SOP (Post-DpnI Treatment)
To join a linearized vector and a
PCR-amplified insert using homologous recombination via the In-Fusion® HD
Cloning Kit.
Reagents and
Equipment Required
-
DpnI-treated, PCR-linearized
vector (purified)
-
PCR-amplified insert (with
15-bp overlaps)
-
In-Fusion HD Enzyme Premix
-
Nuclease-free water
-
Ice, microcentrifuge tubes
-
High-efficiency competent cells
(e.g., NEB 5-alpha)
-
LB agar plates with appropriate
antibiotic
-
Thermocycler
Steps
1. Quantify
DNA
-
Use Nanodrop or Qubit to
measure concentration.
-
Use ~50–100 ng of vector and
insert at a molar ratio of 2:1 (insert:vector).
2. In-Fusion
Reaction Setup (10 µL total)
|
Component |
Volume |
|
Purified linearized vector (50–100 ng) |
X µL |
|
Purified insert DNA (molar ratio 2:1) |
Y µL |
|
In-Fusion HD Enzyme Premix (5×) |
2 µL |
|
Nuclease-Free Water |
Up to 10 µL |
-
Mix gently and briefly spin
down.
3. Incubation
-
Incubate at 50°C for 15
minutes.
-
Place on ice immediately after
incubation.
4.
Transformation
-
Thaw competent E. coli cells
(e.g., NEB 5-alpha) on ice.
-
Add 5 µL of In-Fusion reaction
to 50 µL of competent cells.
-
Gently flick to mix and
incubate on ice for 30 minutes.
-
Plate on LB agar plates with
appropriate antibiotics.
-
Incubate overnight at 37°C.
5. Colony
Screening
-
Pick colonies the next day for
colony PCR or plasmid mini prep + sequencing.