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pLUG-Prime® TA-cloning Vector Kit II

Cat.No	Capacity	Inquire
11063	20 rxn.

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Description
TroubleShooting Guide
Related products
Protocol
MSDS

PRODUCT INFORMATION

Description

pLUG-Prime® TA-cloning Vector Kit II offers a quick, reliable and efficient method for cloning a variety of DNA sequence.

This kit makes it possible to separate insert DNA from vector using Eco RI restriction enzyme.

One of the main advantages of the pLUG-Prime® TA-cloning Vector Kit II is that it has more accurate results due to accepting a wide range of inserts with different sizes. The kit provides two types of ligation buffers provided for your convenience.

pLUG-Prime® TA-cloning Vector Kit II contain several engineered restriction-enzyme recognition sites around the TA-cloning site allowing easy restriction analysis of recombinant plasmids or re-cloning to another vector.

Especially, the restriction-enzyme recognition sites of pLUG-Prime® TA-cloning Vector Kit II around the TA-cloning site are more general and simple, which is useful in downstream application such as re-cloning to another vector.

Characteristics

• High cloning efficiency

• High percentage of true white colony

• Credible blue/white colony selection

• Rapid procedure (rapid ligation)

• Allowing easy re-cloning to another vector

• Allowing convenient sequencing (M13 F/R priming sites)

Application

• TA-cloning

• Accepts terminal 3’-dA nucleotides overhang PCR products

• Transform ligation product (purified/unpurified) into competent cells

• LacZ complementation for blue/white screening

• Ampicillin marker for antibiotic selection

No.	Kit Contents	Unit
1	TA-Cloning Vector II (20 reactions)	25 ng/µl X 40 µl
2	Control insert DNA	10 ng/µl X 10 µl
3	T4 DNA ligase	2 U/µl X 20 µl
4	10X Ligation Buffer A	50 µl
5	10X Ligation Buffer B	50 µl
6	Forward Primer (M13-F)	10 µM X 50 µl
7	Reverse Primer (M13-R)	10 µM X 50 µl
8	Manual	1 ea

Kit Contents

No. Kit Contents Unit

1 TA-Cloning Vector II (20 reactions) 25 ng/µl X 40 µl

2 Control insert DNA 10 ng/µl X 10 µl

3 T4 DNA ligase 2 U/µl X 20 µl

4 10X Ligation Buffer A 50 µl

5 10X Ligation Buffer B 50 µl

6 Forward Primer (M13-F) 10 µM X 50 µl

7 Reverse Primer (M13-R) 10 µM X 50 µl

8 Manual 1 ea

Storage

Should be stored –20°C. Always avoid multiple freeze-thaw cycles or exposure to frequent temperature changes. These fluctuations can greatly alter stability of product.

Technical Data

Map & Multiple cloning site of the pLUG-Prime® TA-cloning vector Kit

Figure 1 : Map and sequence reference points of the pLUG-Prime® TA-Cloning Vector II ( β-Coronavirus )

* Before the insert is incorporated into pLUG-Prime® TA-Cloning Vector II , there is only one Hind Ⅲ site and no Bgl Ⅱ site. After the incorporation, the T and A nucleotide on the insert will complement with the sequence on the vector and generate these two new sites. This merit of pLUG-Prime® TA-Cloning Vector II makes cloning more economical and convenient.

Figure 2 : Multiple cloning site sequence of the pLUG-Prime® TA-Cloning Vector II

Figure 3 : The gel analysis of the PCR products ligated by pLUG-Prime® TA-Cloning Vector Kit II

TroubleShooting Guide

QNo colony was obtained from transformation?

A- Bacteria were not competent.
→ Check the transformation efficiency of competent cells used. Vortex the ligation buffer vigorously before use. We recommend using highly competent cells (≥1.0×108 cfu/μg circular plasmid DNA).

- Incorrect transformation procedure.
→ Make sure that the appropriate transformation procedure was used.

- Use of incorrect antibiotic on plates.
→ Agar plates should include ampicillin; Check the ampicillin.

Q Low number of white colonies?

A - A-tailing of the PCR product was not efficient.
→ Verify that PCR amplification was performed using Taq DNA polymerase; Use fresh PCR products. Efficiency may be reduced within a day because of loss of A overhang of PCR products.

- Insert to vector ratio was too low.
→ Use the 5-10-fold molar excess of PCR product over TA-cloning vector.

- Ligation mixture used in transformation was insufficient.
→ Increase the amount of the ligation mixture added to the transformation reaction or increase the amount of the transformants plated.

- Presence of inhibitor in PCR mixture.
→ The high salt content of PCR reactions can inhibit ligation and transformation. Though your PCR product is shown as single band, purify the PCR product prior to ligation.

- Overexposure of PCR product to UV-light.
→ If the PCR product is gel-purified, the PCR product should be exposed to long-wave UV-light for as short a time as possible. Overexposure of PCR product to UV-light will lead to the formation of pyrimidine dimers that can not be ligated efficiently.

Q White colonies did not have insert?

A - Cells transformed with residual plasmid DNA from PCR reaction.
→ If the PCR template was plasmid DNA containing the resistance gene for the antibiotic used for colony selection, colonies may be shown on the following transformation. In that case, PCR product should be gel-purified prior to ligation reaction.

- Non-specific PCR products or primer dimers cloned into TA-cloning vector.
→ Gel-purify the PCR product prior to performing the TA-cloning reaction; Verify that the PCR primer design and quality.

Q Most colonies were blue or light blue?

A - The insert does not interrupt the reading-frame of the lacZ gene.
→ If the insert is small (<300bp) and the number of its bases including the 3’-end A-overhang is multiple of 3, the recombinant colony may be light blue.

- Inappropriate bacterial strain was used for blue/white colony selection.
→ Ensure that the bacterial strain used for transformation carries lacZ mutation. Phenotype of colony is always blue which is in lacZ+ strains.

- Self-ligation of TA cloning vector.
→ Frequent freezing/thawing may induce loss of 3’-end T-overhang in the TA-cloning vector. Loss of the T-overhangs is the cause of the ligation of vector itself in blunt-end and the colony is shown up as blue; Nuclease may degrade the T-overhang in TA-cloning vector. Use only the provided distilled water and ligation buffer in the ligation reaction.

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