|Key Features:|| |
• One step: combines circular vector linearization and homology-based assembly.
• Rapid: saves 2-3 hours by eliminating vector digestion and gel purification steps.
• Efficient: Typically >95% of colonies bear the correctly inserted DNA fragment.
• Large scale: accommodates up to 500 ng plasmid DNA in one reaction, resulting in more colonies.
• Cost-effective: eliminates vector digestion, gel electrophoresis and purification costs, saving up to 50%.
|Description: ||ExSembly™ Cloning Master Mix is a simple, fast, and high efficient product which enables directional insertion of any amplified DNA product into CIRCULAR vector. The ExSembly™ technology completely eliminates the time consuming step of preparing linear vector. The innovative buffer system in our master mix allows restriction enzymes to efficiently digest the circular DNA, and maintain high exonuclease and polymerase activity to enable the assembly to occur. This technology takes advantage of the fact that once the vector DNA successfully assembles with insert DNA, the restriction sites in the vector disappear. The restriction enzymes remove background negative clones by completely digesting unassembled vector DNA. One can conveniently use a relatively large amount of vector DNA, up to 500 ng, and obtain high number of positive clones, which consequently increase success rate.|
• Fast Cloning
• High-throughput Cloning
• Seamless Assembly
• Site-directed Mutagenesis
|Component:||1. 2× ExSembly™ Cloning Master Mix: 100 µl|
2. Positive control: 50 µl
|Storage:||All components should be stored at -20°C; Avoid repeated freezing and thawing.|
Choose one (or more) restriction enzyme(s*) that linearize the vector and DO NOT cut within the fragment(s) to be inserted. For single fragment insertion, using PCR, add 20 bp homology upstream of the unique restriction site in the vector to the 5’ end of the fragment, and 20 bp of vector homology downstream of the restriction site to the 3' end.
Restriction enzyme compatibility
ORFs of eGFP or human SRSF3 were PCR-amplified and assembled into vectors (pQE-80L, pProex-hta, pRSET-B) using ExSembly™ Master Mix. ~200 ng vector DNA was used in each ExSembly™reaction. ExSembly™products were purified using a DNA mini-spin column and transformed to DH10B electrocompetent cells and plated on LB/Ampicillin plates. Forty colonies were picked from each reaction to perform colony PCR screening. Percent positive clones are shown in Figure 1 for each of 15 common restriction enzymes.
Comparison between ExSembly™ and Competitor N
Competitor N Experiment 1: 120 ng gel-purified vector + 360 ng insert (Figure A); Competitor N Experiment 2: 300 ng column purified vector + 300 ng insert (Figure B)
ExSembly Experiment 1: 200 ng vector + 360 ng insert (Figure C); ExSembly Experiment 2: 600 ng vector + 360 ng insert (Figure D)
"I have been using the ExSembly™ Cloning Master Mix for two years and have had great success in cloning a wide range of DNA fragments using this mix. The product is extremely easy to use and makes cloning a very quick process. It is especially ideal to clone multiple DNA fragments simultaneously into a vector. Compared to other approaches and commercial kits I have used in the past; this product's efficiency is very impressive. I highly recommend the ExSembly™ Cloning Mix."
"ExSembly™ Cloning Master Mix works very well in my hands for almost two years. It has similar or higher efficiency compared with other assembly kits!"