Invitrogen™ TA Cloning™ Kit, Dual Promoter, with pCR™II Vector, without competent cells

Catalog No.	No. of Reactions
K207040	40 Reactions
K207020	20 Reactions

Catalog No. K207040 Supplier Invitrogen™ Supplier No. K207040

Description

Provides quick, one-step cloning strategy for directly inserting Taq-amplified PCR product into plasmid vector

The T7 and Sp6 promoters of the pCRII vector allow in vitro transcription of the insert to produce sense or anti-sense products. The TA Cloning Kit Dual Promoter uses the pCRII 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.

• Fast and 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 PCR product
• Streamlined: does not require use of PCR primers that contain restriction sites
• 3'-T overhangs for direct ligation of Taq-amplified PCR products
• T7 and Sp6 promoters for in vitro RNA transcription and sequencing
• Versatile polylinker with flanking EcoR I sites for easy excision of inserts
• M13 forward and reverse primer sites for sequencing
• Cloning Method: Restriction Enzyme/MCS

• How TA Cloning Works
• Taq polymerase has non-template-dependent activity that adds single deoxyadenosine (A) to 3' ends of PCR products
• Linearized vector supplied in this kit has single 3' deoxythymidine (T) residues
• Allows PCR inserts to ligate efficiently with vector
• Kit Configurations
• TA Cloning Kit is offered in a variety of configurations: with One Shot INVF' Chemically Competent E. coli (K2050-01 and K2050-40), One Shot TOP10F' Chemically Competent E. coli (K2060-01 and K2060-40), and without competent cells (K2750-20 and K2750-40) in 20- and 40-reaction kit sizes

Cloning, PCR Cloning

Specifications

• Bacterial or Yeast Strain: Not Included
• Cloning Method: TA Cloning
• Content And Storage: This kit contains linearized pCR™II 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.
• Format: Kit
• For Use With (Application): PCR Cloning
• No. of Reactions: 40 Reactions
• Product Line: TA Cloning
• Product Type: Cloning Kit
• Promoter: T7, SP6
• Quantity: 40 rxns
• Vector: pCRII

Frequently Asked Questions (FAQs)

How do you recommend that I prepare my DNA for successful electroporation of E. coli?

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.

Have you compared in vitro transcription levels between SP6 and T7 promoters in your pCRII vectors?

No, we have not done in-house comparisons of transcription levels. It is widely known though that T7 polymerase produces more RNA than SP6 (on the order of 10-fold higher).

When should DMSO, formamide, glycerol and other cosolvents be used in PCR?

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.

For Research Use Only. Not for use in diagnostic procedures.