TOPO™ TA Cloning™ Kit, Dual Promoter, with pCR™II-TOPO™ Vector and One Shot™ Mach1™ T1 Phage-Resistant Chemically Competent E. coli
TOPO&trade; TA Cloning&trade; Kit, Dual Promoter, with pCR&trade;II-TOPO&trade; Vector and One Shot&trade; Mach1&trade; T1 Phage-Resistant Chemically Competent <i>E. coli</i>
Invitrogen™

TOPO™ TA Cloning™ Kit, Dual Promoter, with pCR™II-TOPO™ Vector and One Shot™ Mach1™ T1 Phage-Resistant Chemically Competent E. coli

TOPO™ TA Cloning™ Kits are designed for cloning PCR products directly from a PCR reaction in just 5 minutes (1).Read more
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Catalog number K461020
Price (USD)
744.00
Each
Add to cart
Price (USD)
744.00
Each
Add to cart
TOPO™ TA Cloning™ Kits are designed for cloning PCR products directly from a PCR reaction in just 5 minutes (1). They use a pCR™-TOPO™ Vector with covalently bound topoisomerase I for fast cloning and recombinants. pCR™-TOPO™ Vectors include:

• 3´-T overhangs for direct ligation of Taq-amplified PCR products
• Choice of T7 (pCR™2.1-TOPO™) or T7 and SP6 (pCR™II-TOPO™) promoters for in vitro RNA transcription and sequencing. Each vector also contains M13 forward and reverse primer sites for sequencing.
• EcoR I sites flanking the PCR product insertion site for easy excision of inserts
• Kanamycin and ampicillin resistance genes for your choice of selection in E. coli
• Easy blue/white colony screening for selection of recombinants

TOPO™ TA Cloning™ Kits are also available in combo kits combined with a PureLink™ Quick Plasmid Miniprep Kit (50 preps) for fast plasmid purification of your TOPO™-cloned inserts for downstream analysis.
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Bacterial or Yeast StrainMach1™
Cell TypeChemically Competent
Cloning MethodTOPO™-TA
FormatKit
For Use With (Application)Chromatin Biology
Product LineOne Shot™
Product TypeCloning Kit
PromoterT7, SP6
Quantity25 Reactions
VectorTOPO-TA Cloning Vectors
Unit SizeEach
Contents & Storage
Box 1:
• Topoisomerase I-activated pCR™II-TOPO™ vector
• PCR buffer
• Salt solution
• dNTPs
• Control template
• M13 forward and reverse primers
• Control PCR primers
• Sterile water

Store at -5 to -30°C.
All reagents are stable for 6 months when properly stored.

Box 2:
• One Shot™ Chemically Competent or Electrocomp™ E. coli
• S.O.C. medium
• Supercoiled pUC19 control plasmid.

Store at -68 to -85°C.

Frequently asked questions (FAQs)

Can I store my competent E. coli in liquid nitrogen?

We do not recommend storing competent E. coli strains in liquid nitrogen as the extreme temperature can be harmful to the cells. Also, the plastic storage vials are not intended to withstand the extreme temperature and may crack or break.

How should I store my competent E. coli?

We recommend storing our competent E. coli strains at -80°C. Storage at warmer temperatures, even for a brief period of time, will significantly decrease transformation efficiency.

What is the best molar ratio of PCR product:vector to use for TOPO TA cloning? Is there an equation to calculate the quantity to use?

We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1. The quantity used in a TOPO cloning reaction is typically 5-10 ng of a 2 kb PCR product.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 (insert:vector ratio)

What is the best ratio of insert:vector to use for cloning? Is there an equation to calculate this?

The optimal ratio is 1:1 insert to vector. Optimization can be done using a ratio of 0.5-2 molecules of insert for every molecule of the vector.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio

Does Platinum Taq DNA Polymerase High Fidelity enzyme mix leave 3' A-overhangs on the PCR product for subsequent cloning into a TOPO TA or original TA vector?

Yes, the enzyme mix leaves 3' A-overhangs on a portion of the PCR products. However, the cloning efficiency is greatly decreased compared to that obtained with Taq polymerase alone. It is recommended to add 3' A-overhangs to the product for TA cloning.

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