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One-step Fusion Cloning Kit
Description
1. Preparation of Linearized Vector
The one-step Fusion Cloning Kit is designed for fast, directional
cloning of DNA fragment into any vector at any site. It can simplify and
accelerate your experiments without requiring PCR product cutting
with restriction enzymes allowing flexible options for restriction
enzyme sites when linearizing the vector. Additionally, the linearized
vector and PCR product can be cyclized in only 30 minutes at 37 ℃.
Compared to the conventional cloning method, the 15 base tract is
added at the ends of PCR primers corresponding to the ends of the
linearized vector.
The vector linearization is necessary for proper DNA fusion reaction
and can be generated using restriction enzymes or long and accurate
PCR. In order to increase fusion efficiency and decrease background,
we recommend linearizing the vector by double restriction enzyme
digestion for 2 hours or overnight if desired.
Components
B22611 (40 rxns)
Contents
Fusion Enzyme (µL)
5 x Fusion Buffer (µL)
Linearized Control Vector - 2.7kb (µL)
Control DNA Fragment - 500bp (µL)
User Manual
B22612 (100 rxns)
40
80
5
5
100
Yes
Yes
After digestion, purify the linearized vector using a DNA gel extraction
kit for high purification efficiency to increase the chances of a
successful fusion reaction.
2. PCR Primer Design
Example of primers designed for Fusion cloning
PCR primers must contain 15-20 bp sequences that are homologous
to the end of the vector. Consider the following parameters:
200
5
5
3. Fusion Cloning Reaction
Storage
One-step Fusion Cloning Kit should be stored at -20 °C.
Protocol
Overview
1.Generate a linearized vector
2.PCR amplification of the insert
Set up the following fusion cloning reaction by mixing the following
reagents. Spin down briefly after each addition to collect the reagents
at the bottom of the tube.
Linearized Vector
Insert
Fusion Enzyme
5 x Fusion Buffer
ddH2O
20-100 ng
10-100 ng
1 µL
2 µL
Variable
10 µL
RE digestion, PCR, ...
Gene-specific primers with 15bp
extension homologous to vector ends
2:1 molar ratio of insert: vector presents the highest fusion efficiency.
The optimal quantity of vector and insert is as follows:
Linearized Vector: [0.01 * the length of vector (bp) ] ng
Insert: [0.02 * the length of insert (bp) ] ng
Positive Control Reaction
3. Set up the fusion cloning reaction
One-step Fusion Cloning Kit
Incubation at 37°C for 30 minutes
4.Transformation
Clone screening
Linearized Control Vector (2.7kb)
Control DNA Fragment (500bp)
Fusion Enzyme
5 x Fusion Buffer
ddH2O
0.5 µL
0.5 µL
1 µL
2 µL
6 µL
10 µL
Fusion Reaction Conditions: Incubate at 37 °C for 30 minutes
(optimal time), then place on ice. Immediately proceed to the
transformation step or store the reaction tubes at –20°C.
Order & Inquiry
Order & Inquiry
Tel: (713)732-2181
Fax: +1-866-747-4781
E-mail: [email protected]
Tel: +49-89-46148500
Fax: +49-89-461485022
E-mail: [email protected]
4. Transformation
1. Add 100 µL competent cells to the fusion reaction mixture
2. Mix gently and Incubate on ice for 30 minutes
3. Heat shock at 42°C for 60 seconds
4. Transfer on ice for 2 minutes
5. Add 500 µL SOC or LB antibiotic free medium
6. Incubate at 37°C for an hour
7. Spread 100 µL culture to LB plate containing the appropriate
antibiotic.
8. Incubate the plate at 37°C overnight
We strongly recommend the use of competent cells with a
transformation efficiency ≥ 1 x 108 cfu/µg. If the efficiency is lower
than the recommended amount, centrifuge the culture, resuspend the
pellet in 100 µL medium, and then, spread the entire suspension.
About 16 hours later, pick individual monoclones from the LB plate
and incubate with liquid LB medium in 37°C shaker overnight. The
next day, to determine the presence of the insert, perform PCR
screening and plasmid extraction.
Troubleshooting
Please review the following for trouble-shooting options.
Alternatively, feel free to contact Biotool technical support directly
with any technical difficulties.
Problem
No or few
colonies
obtained
Large numbers
of colonies
contain
no insert
Clones contain
incorrect insert
Potential Cause(s)
Suggestion(s)
Primer sequences
are incorrect
Check primer sequences
to ensure
the overlap correct
PCR product is not pure
or the DNA
concentration is low
Use a different method to
purify your PCR product again
Transformation contained
too much fusion reaction
mixture
Make sure the volume of
reaction mixture is no more
than 10 % of competent cells
Repeat transformation
experiment with efficient
Low transformation
competent cells
efficiency of competent cells
(efficiency ≥ 1 x 108 cfu/ug)
Wrong antibiotic used or
too much antibiotic
in the LB medium
Choose plates with the
appropriate concentration of
the correct antibiotic
The linearization of
vector is incomplete
Repeat digestion of
vector with more time
and gel purify
Contamination of
plasmid in
PCR fragment
When amplifying from a
plasmid, the PCR
product must be purified
LB plates are too old
or the antibiotic
resistance is incorrect
Make sure that your LB
plates are fresh
and the antibiotic
resistance is correct
PCR products contain
nonspecifically
amplified fragments
Optimize the PCR
reaction to improve the
specificity or screen
more colonies
for the correct clones
Order & Inquiry
Order & Inquiry
Tel: (713)732-2181
Fax: +1-866-747-4781
E-mail: [email protected]
Tel: +49-89-46148500
Fax: +49-89-461485022
E-mail: [email protected]