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TA Cloning™ Kit, with pCR™2.1 Vector, without competent cells
The TA Cloning™ Kit with pCR™2.1 vector provides a quick, one-step cloning strategy for directly inserting a Taq-amplified PCR productRead more
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| Catalog Number | No. of Reactions |
|---|---|
| K202020 | 20 Reactions |
| K202040 | 40 Reactions |
Catalog number K202020
Price (EUR)
345,00
Each
-
No. of Reactions:
20 Reactions
Price (EUR)
345,00
Each
The TA Cloning™ Kit with pCR™2.1 vector provides a quick, one-step cloning strategy for directly inserting a Taq-amplified PCR product into a plasmid vector. The TA Cloning™ Kit uses the pCR™2.1 cloning vector and ExpressLink™ T4 DNA Ligase to generate a ligation product in a fifteen-minute, room-temperature ligation step. Reactions typically yield >80% recombinants containing inserts.
Features of the TA Cloning™ Kit with pCR™2.1 vector:
• Fast & convenient—15-minute, room-temperature ligation
• Efficient—blue/white screening and >80% clones with correct insert
• Flexible—choice of kanamycin or ampicillin resistance for flexible antibiotic selection
• Hassle-free—eliminates any enzymatic modifications of the PCR product
• Streamlined—does not require the use of PCR primers that contain restriction sites
The pCR™2.1 vector provides:
• 3'-T overhangs for direct ligation of Taq-amplified PCR products
• T7 promoter for in vitro RNA transcription and sequencing
• A versatile polylinker with flanking EcoR I sites for easy excision of inserts
• M13 forward and reverse primer sites for sequencing
How TA Cloning™ Works
Taq polymerase has a non-template-dependent activity that adds a single deoxyadenosine (A) to the 3' ends of PCR products. The linearized vector supplied in this kit has single 3' deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.
Kit Configurations
The TA Cloning™ Kit is offered in a variety of configurations: without competent cells (K2020-20 and K2020-40), with One Shot™ INVF' Chemically Competent E. coli (K2000-01 and K2000-40), with One Shot™ TOP10F' Chemically Competent E. coli (K2030-01 and K2030-40), and with One Shot™ TOP10 Chemically Competent E. coli (K2040-01 and K2040-40) in 20- and 40-reaction kit sizes.
Features of the TA Cloning™ Kit with pCR™2.1 vector:
• Fast & convenient—15-minute, room-temperature ligation
• Efficient—blue/white screening and >80% clones with correct insert
• Flexible—choice of kanamycin or ampicillin resistance for flexible antibiotic selection
• Hassle-free—eliminates any enzymatic modifications of the PCR product
• Streamlined—does not require the use of PCR primers that contain restriction sites
The pCR™2.1 vector provides:
• 3'-T overhangs for direct ligation of Taq-amplified PCR products
• T7 promoter for in vitro RNA transcription and sequencing
• A versatile polylinker with flanking EcoR I sites for easy excision of inserts
• M13 forward and reverse primer sites for sequencing
How TA Cloning™ Works
Taq polymerase has a non-template-dependent activity that adds a single deoxyadenosine (A) to the 3' ends of PCR products. The linearized vector supplied in this kit has single 3' deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.
Kit Configurations
The TA Cloning™ Kit is offered in a variety of configurations: without competent cells (K2020-20 and K2020-40), with One Shot™ INVF' Chemically Competent E. coli (K2000-01 and K2000-40), with One Shot™ TOP10F' Chemically Competent E. coli (K2030-01 and K2030-40), and with One Shot™ TOP10 Chemically Competent E. coli (K2040-01 and K2040-40) in 20- and 40-reaction kit sizes.
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Bacterial or Yeast StrainNot Included
Cloning MethodTA Cloning
For Use With (Application)PCR Cloning
No. of Reactions20 Reactions
Product LineTA Cloning™
Product TypeCloning Kit
PromoterT7
Quantity20 reactions
VectorpCR2.1
FormatKit
Unit SizeEach
Contents & Storage
TA Cloning™ kits contain linearized pCR™2.1 vector, ExpressLink™ T4 DNA ligase, 5X ExpressLink™ T4 DNA ligation buffer, dNTPs, 10X PCR buffer, sterile water, and controls.
Store all components at -20°C. All reagents are guaranteed stable for 6 months when properly stored.
Store all components at -20°C. All reagents are guaranteed stable for 6 months when properly stored.
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Frequently asked questions (FAQs)
For best results, DNA used in electroporation must have a very low ionic strength and a high resistance. A high-salt DNA sample may be purified by either ethanol precipitation or dialysis. The following suggested protocols are for ligation reactions of 20ul. The volumes may be adjusted to suit the amount being prepared. Purifying DNA by Precipitation: Add 5 to 10 ug of tRNA to a 20ul ligation reaction. Adjust the solution to 2.5 M in ammonium acetate using a 7.5 M ammonium acetate stock solution. Mix well. Add two volumes of 100 % ethanol. Centrifuge at 12,000 x g for 15 min at 4C. Remove the supernatant with a micropipet. Wash the pellet with 60ul of 70% ethanol. Centrifuge at 12,000 x g for 15 min at room temperature. Remove the supernatant with a micropipet. Air dry the pellet. Resuspend the DNA in 0.5X TE buffer [5 mM Tris-HCl, 0.5 mM EDTA (pH 7.5)] to a concentration of 10 ng/ul of DNA. Use 1 ul per transformation of 20 ul of cell suspension. Purifying DNA by Microdialysis: Float a Millipore filter, type VS 0.025 um, on a pool of 0.5X TE buffer (or 10% glycerol) in a small plastic container. Place 20ul of the DNA solution as a drop on top of the filter. Incubate at room temperature for several hours. Withdraw the DNA drop from the filter and place it in a polypropylene microcentrifuge tube. Use 1ul of this DNA for each electrotransformation reaction.
Cosolvents may be used when there is a failure of amplification, either because the template contains stable hairpin-loops or the region of amplification is GC-rich. Keep in mind that all of these cosolvents have the effect of lowering enzyme activity, which will decrease amplification yield. For more information see P Landre et al (1995). The use of co-solvents to enhance amplification by the polymerase chain reaction. In: PCR Strategies, edited by MA Innis, DH Gelfand, JJ Sninsky. Academic Press, San Diego, CA, pp. 3-16.
Additionally, when amplifying very long PCR fragments (greater than 5 kb) the use of cosolvents is often recommended to help compensate for the increased melting temperature of these fragments.
Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.