DNA Assembly Cloning

Assay Genie DNA Assembly Cloning Kits

SKU Product Size

MORV0102/MORV0103

25 rxn/50 rxn

GenieClone DNA Assembly Cloning Kit

The GenieClone cloning technology is simple, fast, and highly efficient. It enables directional insertion of any amplified DNA product into any linearized vector at any site.

Firstly, the vector is first linearized at the cloning site. A small overlapping sequence at each end of the cloning site is added to the insert through PCR. The insert and the linearized vector both with overlapping 5’- and 3’ (15 bp - 20 bp) sequences are mixed in an appropriate ratio and incubated with GenieClone Recombinase at 50℃ for 5 - 15 min before transformation of competent cells for successful DNA cloning and assembly.

GenieClone UItra One Step Cloning kit allows for the highly efficient cloning and assembly of 1-5 DNA fragments in as little as 15 minutes into any vector at any site. This system utilizes ligation-independent technology and a novel GenieClone recombinase to significantly reduce self-ligating colonies. The enhanced GenieClone Recombinase and highly optimized buffer included in the 2x GenieClone Mix significantly improve the recombination efficiency and the tolerance to impurities. The pCE-ONE vector is compatible with most PCR products, enabling the specific PCR fragments to be used directly for recombination without any pre-treatment.

Figure: Mechanism of GenieClone Simple Homologous Recombination

GenieClone DNA Assembly Cloning Kit - Applications

  • Fast Cloning
  • High-throughput Cloning
  • Seamless Assembly
  • Site-specific Mutagenesis

DNA Assembly Cloning Tips

1. Place the recombination products on ice and transform it to competent cells directly.
Commercial super competent cells (transformation efficiency over 10 8 cfu/μg) are highly recommended. The volume of transformation products should not be more than 1/10 of the volume of competent cells.

2. GenieClone UItra One Step Cloning Kit is applicable for efficient clone of fragments of 50 bp - 10 kb.

3. Usages of inserts and vectors
Cloning fewer or short fragments (< 5 kb):

  • Linearized vectors, prepared by restriction digestion, can be directly used for recombination after inactivating the restriction enzymes. Inactivation is available for most restriction enzymes, please refer to instruction of specific inactivation methods.
  • For linearized vectors prepared by reverse PCR, if the amplification templates are pre-linearized and PCR products show singer band, the PCR products can be used directly for recombination without purification.
  • For inserts, if the yield and amplification specificity of the PCR products is confirmed by agarose electrophoresis and the templates are not circular plasmids which share the same antibiotic resistance with the cloning vector, high specific PCR products can be directly used. For recombination without further purification, please refer to Table 1 / Table 2 for the usages of linearized vectors and inserts prepared in different ways.

Table 1: Usage of Linearized Vectors

Method of Linearization Template Type Fast Protocol Standard Protocol

Digestion

Circular Plasmid

Use directly after inactivating restriction enzymes

Gel Recovery

Reverse PCR

Specific Amplification

Circular Plasmid

Use directly after Dpn I digestion

(degrade the PCR template)

Gel recovery or gel
recovery after Dpn I digestion

Reverse PCR

Specific Amplification

Pre-linearized Plasmid, Genomic DNA, cDNA

Use directly

Gel recovery

Reverse PCR

Non-Specific Amplification

Gel recovery

Gel recovery

Gel recovery

Table 2: Usage of Amplified Inserts

Amplification Specificity Template Type Fast Protocol Standard Protocol

Specific Amplification

Circular plasmids sharing the same antibiotic resistance with the cloning vector

Use directly after Dpn I digestion

Gel recovery or gel recovery after Dpn I digestion

Specific Amplification

Pre-linearized Plasmid, Genomic DNA, cDNA

Use directly

Gel recovery

Non-Specific Amplification

Gel recovery

Gel recovery

Gel recovery

  • When using enzyme- digestion products or amplified products directly for recombination, the volume should be ≤ 2μl (≤ 1/5 of the total volume of recombination reaction system).
  • After Dpn I digestion, the amplified inserts should be incubated at 85℃ for 20 min to deactivate Dpn I , so as to prevent cloning vectors from degradation when recombination.
  • Cloning multiple fragments (4 - 5) or large fragment (> 5 kb). It is recommended to purify the linearized vectors and amplified inserts with high quality gel DNA recovery kit before recombination, so as to improve the DNA purity and eliminate residual circular vectors.

DNA Assembly Cloning Troubleshooting

Problem Corrective Action

Primers design notes

The linearized vector can be obtained by double digestion, single digestion and reverse PCR, among which, double digestion is recommended.

Three parts of primers: homologous sequences (15 bp – 20 bp, exclude restriction sites and base residues, the content of GC is 40% - 60%) + restriction sites (optional according to experiment need) + specific primers (when calculating the Tm of primers, the homologous sequence should be excluded).

Few clones or no clones formed on the plate

Improper primer design: primer includes 15 bp - 20 bp homologous sequences (exclude restriction sites); the content of GC is 40% - 60%.

The amount of linearized vectors and amplified inserts are too low/high in the recombination reaction or the ratio of fragments is not appropriate. Please use the amount and radio as specification recommended.

Contamination in vector and insert inhibits the recombination: The total volume of unpurified vector and insert digested should be ≤ 2μl (≤ 1/5 of the total volume of recombination reaction system). Gel extraction purification is recommended to purify the vector and insert. It is recommended to dissolve the purified DNA in ddH2O.

The low efficiency of the competent cell: Make sure the transformation efficiency of competent cells is >108 cfu/μg. Transform 1 ng vector and pick 1/10 of that to transform, and there are 1000 colonies growing on the plate. Then, the transformation efficiency of competent cells can be estimated as 108 cfu/μg. The volume of transformation products should not be more than 1/10 of the volume of competent cells. Choose competent cells used for cloning (such as DH5α/XL10) not those engineered for protein expression.

Incorrect/none inserts found in the colony plasmids

Non-specific amplification is mixed with target inserts: Optimize the PCR reaction system to improve the amplification specificity; purify the PCR products with a gel recovery kit; select more colonies for verification.

Incomplete linearization of the vector: Approaches to overcome such situation include using negative controls to confirm the complete linearization of vectors, improving the amount of restriction endonuclease, prolonging the digesting time, and purifying the digesting products before the recombination reaction.

Plasmids with the same resistance with vectors mixed in reaction system: When the PCR templates for amplification of vectors or inserts are circular plasmids, digesting the amplification products with Dpn I or purifying them by gel recovery can both effectively reduce or even eliminate the residues of cyclic plasmid templates.

No electrophoretic bands in colony PCR

Improper primer: it is recommended to use at least one common sequencing primer of the vector.

Inappropriate PCR system or program: No bands of targets or empty plasmids. It is recommended to optimize the PCR reaction system or program; extract plasmids as PCR templates or use enzyme digestion for confirmation.

Unsuccessful recombination: There is only the band of empty plasmid after colony PCR, which indicates the unsuccessful recombination and incomplete linearization of the vector. One of the approaches to overcome such situation is to optimize the enzyme digestion system.