GenieClone 5 Minute TA ZERO PCR Cloning Kit

SKU:
MORV0009
€150

Description

Introduction

GenieClone 5 minute TA Cloning Kit is a second generation TOPO cloning kit that contains a second generation Topoisomerase, a vector containing the suicide gene ccdB and a blunt end factor. Combining with the optimal buffer, the second generation of Topoisomerase provides a highly efficient, 5 minute, one-step cloning strategy at room temperature.

This product using a vector containing the suicide gene ccdB, when the insert is successfully ligated to the vector, the correct expression of ccdB is destroyed, and the host cell can grow normally, otherwise the host cell cannot grow normally, thereby achieving “zero” background. Containing a blunt end factor, 5minTM TA/Blunt-Zero Cloning Kit is compatible with both TA clones and blunt clones.

Kit Components

Components MORV0009 - 25 rxn MORV0009 - 50 rxn

GenieClone 5 Minute TA ZERO PCR Cloning mixa

25 μl

2 x 25 μl

500 bp Control insert (20 ng/μl)

5 μl

10 μl

M13 Primer Mix (10 μM)b

200 μl

400 μl

a. Contains Topoisomerase and pCE2 TA/Blunt-zero Vector (double resistance: Amp , Kan )
b. SM13 Forward Primer AM13 Reverse Primer

Storage and Shipping

Store at -30℃ to -15℃. Transportation condition is -20℃ to 0℃

Protocol

1. Summary of the Experimental Process

Figure 1: Process Summary of GenieClone 5 Minute TA ZERO PCR Cloning Kit

Figure 1: TA-zero Cloning (Left)

  1. Add the PCR amplification product with 3' end containing an A overhang to the GenieClone 5 Minute TA ZERO PCR Cloning mix and incubate at room temperature for 5 min.
  2. The blunt-end factor in the Cloning Mix removes the A-base at the end of the amplification product to form a blunt-ended product.
  3. The 5'-OH of the blunt-end product attacks the phosphate bond between the TOPO enzyme and the vector. Once the TOPO enzyme is released the vector it forms a circular recombinant with the blunt-ended product.

Figure 1: Blunt-zero Cloning (Right)

  1. PCR Amplification products (blunt ends) from high-fidelity enzymes (such as Assay Genies Fusion Ultra series) are added to the GenieClone 5 Minute TA ZERO PCR Cloning mix and incubated at room temperature for 5 min.
  2. 5'-OH of the blunt-end product attacks the phosphate bond between the TOPO enzyme and the vector. Once the TOPO enzyme is released, the vector forms a circular recombinant with the blunt-ended product.

2. PCR Product Preparation

  1. Primer requirements: the 5' end of the primer must not be phosphorylated.
  2. Enzyme selection: It is recommended to use Genie Fusion polymerases.
  3. PCR Product requirements: Ensure the optimal yield and quality of PCR products via electrophoresis. If the PCR product contains only 1 target band it can be used directly in the cloning reaction. If non-specific bands or primer dimers are detected, perform gel purification & clean-up steps. If the amplification template is plasmid, purification is recommended.

3. Ligation Reaction

Prepare the reaction mixture:

Components Volume

GenieClone 5 Minute TA ZERO PCR Cloning mix

1 μl

Purified PCR Product

1-4 μl

ddH2O

To 5 μl

Mix contents gently by flicking the tube and collect all the liquid at the bottom via low speed centrifuge at room temperature (20 - 37℃) for 5 min. Then place tube on ice.

Recommended reaction conditions

a. The optimum quantity of inserts used = [0.05 × fragment base pairs] ng;
For example, when the insert is 1000 bp, the optimal amount is [0.05×1000] ng or 50 ng in total. Due to the wide range of compatibility of inserts with this product, you can also use the recommended amounts in the table below:

Insert Sizes Recommended Storage

0.5 -1 kb

5 -6 ng

1 – 2 kb

60 – 110 ng

2 – 5 kb

110 – 260 ng

> 5 kb

> 260 ng

b. Reaction Temperature: The kit is compatible with varying reaction temperatures, so the reaction can be performed at room temperature (20 - 37℃) (in a PCR instrument).

c. Reaction Time: Incubate for 5 min.

4. Conversion

This product is compatible with many conventional competent cells (eg. DH5а competent cell; FastT1 competent cell)

*It is recommended to use Fast-T1 competent cell for subsequent transformation experiments. The cells are the fastest growing competent cells (clones can be seen 8h after plating), and the transformation efficiency is high, saving screening time.

5. Positive Clone Identification

a. PCR identification of the bacterial colony: Pick a single colony to 10 μl of ddH2O as a template; 2 × Rapid Taq Master Mix are recommended.

Reaction System:

Components Volume

2x Taq Master Mix

10 μl

M13 Primer Mix

2 μl

Bacterial Solution

2 μl

ddH2O

6 μl

 

Reaction Procedure:

Temperature Time Cycles

95℃

5 min

95℃

15 sec

35 cycles

55℃

30 sec

35 cycles

72℃

60 sec/kb

35 cycles

72℃

10 min

 

b. Enzyme Digestion Analysis:According to the experimental design, select the appropriate restriction endonuclease to identify.

c. Identification of Plasmid Size: Picking a single clone, after plasmid extraction, electrophoresis observation of plasmid size identification.

d. Sequencing Analysis: Directly pick the monoclonal sequencing identification, sequencing primers can choose M13 Forward Primer, M13 Reverse Primer or design it yourself.

Sequence Information of Vector