Isothermal DNA Assembly Kit

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Instructions:

1. Thaw the reaction mix on ice without vortexing, spin down after the thawing if necessary.

2. Prepare the reaction described below.

Reagent	Amount
Reaction Mix	5 µl*
DNA (Vector)	0.02–0.5 pmol
DNA (Insert 1, 2, 3, …)	0.02–0.5 pmol for each insert**
Water (Ultra Pure)	X***
Total Reaction Volume	10 µl

3. Incubate the mix at 50 °C for 15 minutes to 1 hour.

4. Directly add the all reaction mix to the chemically prepared competent cells. For electroporation, add only 0.1µl-0.5µl the mix. The high salt concentration in the reaction may cause arcing (a spark) during electroporation, which can damage both cells and equipment. (Electroporation is not recommended.)

 

*The reaction volume can be increased to 10µl if needed.

**Vector to insert ratio is recommended 1:1 if inserts are bigger than 500bp. For inserts 200 bp<x<500bp length, use 1:3 ratio. For inserts smaller than 200 bp, a 1:6 to 1:12 ratio should be used. Do not use inserts smaller than 100 bp.

***Ultra-pure water is added to bring the total reaction volume to 10 µl.

1. No or low colony yield

• Ensure DNA fragments are clean and free of contaminants like salts, ethanol, or detergents that can inhibit enzymatic activity.

• Verify fragment concentrations; insufficient DNA input may reduce efficiency.

• Confirm that DNA fragments have 20–40 bp overlaps; poor or no homology will prevent assembly.

2. Incorrect or unexpected clones

• Check the sequence and design of overlap regions; mismatches or secondary structures can lead to mis-assembly.

• Sequence errors in primers or templates may produce incorrect junctions—validate all inputs beforehand.

3. High background (non-recombinant colonies)

• Linearize vector completely and remove residual circular plasmid using DpnI or gel purification.

• If blunt-end vectors are used, treat with phosphatase to reduce self-ligation.

4. No growth after electroporation

• Use only 0.1–0.5 µL of assembly reaction for electroporation; higher volumes can cause arcing due to high salt concentration.

• Make sure electrocompetent cells are properly prepared and handled; include a control to test cell competency.

5. Poor efficiency with small inserts

• Use higher vector-to-insert molar ratios for small fragments: 1:6 to 1:12 for inserts <200 bp.

• Avoid using inserts smaller than 100 bp, as assembly efficiency drops significantly.

6. Assembly mix not working

• Avoid repeated freeze–thaw cycles of the isothermal assembly mix; aliquot and store at –20°C.

• Thaw the mix on ice and mix gently before use—do not vortex.

7. Over-incubation of reaction

• Limit incubation to 15–60 minutes at 50°C; longer times may not improve yield and can degrade DNA if contaminants are present.

To ensure efficient and seamless assembly, primers should be designed to introduce 20–40 bp overlapping sequences between adjacent DNA fragments. These overlaps guide the enzymes in the isothermal mix to anneal, chew back, and extend the fragments into a continuous construct.

Overlap Design

• Design 20–40 bp overlaps between the ends of adjacent fragments.

• Overlaps should have a melting temperature (Tm) of 50–60°C.

• Avoid secondary structures, high GC content at the 3’ ends, and long homopolymers within overlaps.

Primer Structure

• Each primer consists of two parts:

5′ Overlap Region (non-annealing): Homologous to the adjacent DNA fragment to be assembled.

3′ Binding Region (annealing): Specific to the template being amplified.

Example:

Forward primer = 5′ [Overlap with Vector or Fragment 1] + [Target-specific binding site] 3′

Reverse primer = 5′ [Overlap with Fragment 3] + [Reverse complement of template] 3′

Length and Tm

Binding region: ~18–25 bp with Tm ~55–65°C

Overlap region: ~20–40 bp with Tm ~50–60°C

Total primer length: 38–65 bp

Below is a list of useful tools for primer design, analysis and melting temperature (Tm) calculation. Please note that these tools are provided by third-party companies that are not affiliated with Synbiotik Co. Synbiotik Co. does not accept any responsibility for the accuracy or outcomes of results generated by these tools;

Benchling
Geneious
IDT OligoAnalyzer
Eurofins Oligo Analysis Tool
NCBI Primer Design and Analysis Tool
PrimerDigital Web Tools for Primer Design and Analysis

For technical support, please contact us through [email protected]