GenieClone DNA Assembly Cloning Kit

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SKU:
MORV0004
€251

Description

ELISA Kit Technical Manual

Product Information

GenieClone DNA Assembly 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.

Utilizing a simple procedure, 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.

 
 

Figure 1: Mechanism of GenieClone Simple Homologous Recombination

Product Components

Components MORV0004- 25 rxn MORV0004 - 50 rxn

2X GenieClone Mix

125 µl

2 x 125 µl

pCE-Zero Vector, Linearized (50 ng/μl)*

25 µl

50 µl

500 bp Control Insert (20 ng/μl)

5 µl

5 µl

*Double-resistance vector, Amp+, Kan+

Storage

All components should be stored at -20℃.
*avoid repeated freezing and thawing.

Applications

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

Materials Needed But Not Supplied

  • PCR templates, primers, linearized vectors.
  • High-fidelity polymerase: Genie Fusion Ultra High-Fidelity DNA Polymerase (Assay Genie # MORV0001) or other equivalent products.
  • Competent cells: chemically competent cells by cloning strains:
    • DH5α competent strain for conventional cloning, applicable to plasmids<15 kb ;
    • XL10 competent strain for long-fragment cloning, applicable to plasmids>10 kb .
  • Other materials: ddH2O, PCR tubes, PCR instrument, etc.
 
 

Figure 2: Primer Design for GenieClone Simple Homologous Recombination

Quick Workflow

1.1 Quantity of Linearized Vectors and Inserts
• Simple homologous recombination and single-fragment homologous recombination:
The optimal mass of vector required = [0.02 × number of base pairs] ng (0.03 pmol)
The optimal mass of insert required = [0.04 × number of base pairs] ng (0.06 pmol)

• Multi-fragment (2 - 5) homologous recombination:
The optimal mass of vector required = [0.02 × number of base pairs] ng (0.03 pmol)
The optimal mass of each insert required= [0.02 × number of base pairs] ng (0.03 pmol)

1.2 Recombination
(a)The amount of DNA can be roughly calculated according to the above formula. Dilute linearized vectors and inserts before recombination to assure the loading accuracy. The volume of each component loaded should be no less than 1 μl.

(b) Prepare the following reaction in ice:

Components Recombination Negative Control - 1 Negative Control - 2 Positive Control

Linearized
Vector

X µl

X µl

0 µl

1 µl

Insert (n≤5)

Y1 + Y2…+Yn µl

0 µl

Y1 + Y2…+Yn µl

1 µl

2X GenieClone
Mix

5 µl

0 µl

0 µl

5 µl

ddH2O

To 10 µl

To 10 µl

To 10 µl

To 10 µl

 

(c) Gently pipette up and down for several times to mix thoroughly (DO NOT VOTEX!). Spin briefly to bring the sample to the bottom of the tube before reaction.

(d) Single-fragment homologous recombination: Incubate at 50℃ for 5 min and chill the tube immediately at 4℃ or on ice.
Multi-fragment homologous recombination: Incubate at 50℃ for 15 min and chill the tube immediately at 4℃ or on ice.

▲Increase the volume of reaction system to 20 µl if the total volume of vector and insert is more than 5µl. For single-fragment homologous recombination, increasing the recombination time to 15min may be helpful to improve the recombination efficiency when the amount of DNA is between 300 ng and 400 ng. For multi-fragments homologous recombination, prolonging the recombination time to 15min, but no more than 1h, can improve the recombination efficiency.

1.3 Transformation

  1. Place the competent cells on ice ( i.e. i.e., DH5α Competent at least >108 cfu/μg).
  2. Pipet 5 - 10µl of the recombination products to 100 µl competent cells, flip the tube for several times to mix thoroughly (DO NOT VOTEX!), and then place the tube still on ice for 30 min.
    *The volume of transformation products should not be more than 1/10 of the volume of competent cells.
  3. Heat-shock the tube at 42℃ for 45sec and then immediately chill on ice for 2-3 min.
  4. Add 900μl of SOC or LB medium (without antibiotics) to the tube. Then, shake at 37℃ for 1h at 200 - 250 rpm.
  5. Preheat the LB plate which contains appropriate selection antibiotic at 37℃.
  6. Centrifuge the culture at 5,000 rpm for 5 min, drop 900 µl of supernatant. Then, re-suspend the pellet with 100 µl of remaining medium and plate it on an agar plate which contains appropriate selection antibiotic.
  7. Incubate at 37℃ for 12 - 16 hours.
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