Preparation of vector/insert for In-fusion cloning via
PCR:
PCR-based vector preparation is used to
linearize and amplify a plasmid backbone with defined ends, often incorporating
overlaps for downstream cloning. This method eliminates the need for
restriction enzymes and offers flexibility in insert design.
Reagents and
Equipment Required
-
Template plasmid DNA
(supercoiled, high-purity)
-
Q5 hotstart
-
primers
-
Nuclease-free water
-
Thermocycler
-
Gel electrophoresis system
-
Agarose, DNA stain (SYBR Safe)
-
DNA gel extraction kit or
column purification kit
-
DpnI enzyme (for template
digestion)
-
spectrophotometer (e.g.,
NanoDrop)
Steps
1. Primer
Design
Design primers that flank the region to be
amplified:
-
Use 20–25 bp that match the
plasmid on each end.
-
For cloning: Add 15–20 bp
overhangs that are homologous to the insert (for Gibson or In-Fusion cloning).
-
Primers should face outward,
amplifying the plasmid backbone minus the insert.
2. PCR Reaction Setup
|
Component |
Volume |
|
Q5 hotstart |
25 µL |
|
Forward primer (10 µM) |
2.5 µL |
|
Reverse primer (10 µM) |
2.5 µL |
|
Template plasmid |
20 ng |
|
Nuclease-free water |
Up to 50 µL |
3. PCR
Conditions (Typical)
|
Step |
Temperature |
Time |
|
Initial denaturation |
98°C |
30 s |
|
35 cycles: |
||
|
– Denaturation |
98°C |
10 s |
|
– Annealing |
55–65°C |
15–30 s |
|
– Extension |
72°C |
30 s/kb |
|
Final extension |
72°C |
5 min |
4. Remove
non-specific PCR Products
-
by agarose gel electrophoresis
and gel cleanup
4. Template
Removal (DpnI Treatment)
-
To remove parental methylated
plasmid template:
-
Add 1 µL DpnI to the PCR
product.
-
Add Rcut smart buffer.
-
Incubate at 37°C for 30–60
minutes.
5.
Purification
-
by agarose gel electrophoresis
and gel cleanup