InsTAclone™ PCR Cloning Kit

- Store at -20°C

Catalog#	Size, concentration	Certificate of Analysis	MSDS

K1213	for 10 react.	K1213
K1214	for 30 react.	K1214

Product information

PCR product cloning procedure

Features

Fast cloning – approximately one hour from PCR completion to cell plating.
High efficiency – more than 90% of the recombinant clones contain the target DNA.
One-step procedure – additional modifications of PCR fragment are not required.
Compatibility – compatible with Taq, Tth, Tfl and other non-proofreading DNA polymerases, as well as with Long PCR Enzyme Mix.
Convenience of pTZ57R/T cloning vector:
– ready-to-use: linearized with Eco32I and 3’-ddT tailed,
– MCS designed for easy mapping and manipulation of the cloned insert,
– blue/white screening,
– M13/pUC primer sites for sequencing or colony PCR screening,
– T7 promoter for in vitro transcription of the insert.

Applications

TA cloning.
Sequencing of cloned insert.
in vitro transcription of insert DNA.

Description

The InsTAclone™ PCR Cloning Kit is a TA system for direct one-step cloning of PCR products with 3'-dA overhangs (1) generated by Taq DNA polymerase and other thermostable DNA polymerases which lack proofreading activity. The high quality ready-to-use TA cloning vector pTZ57R/T (see Fig 1) was prepared by linearizing the pTZ57R plasmid with Eco32I and tailing with single ddT. The 3'-ddT overhangs at both ends of the cloning site prevent recircularization of the vector during ligation, resulting in high cloning yields and low background. To increase the speed, convenience and efficiency of cloning experiment, the InsTAclone™ PCR Cloning Kit has been combined with the unique TransformAid™ Bacterial Transformation Kit – a set of solutions for preparation of chemically competent E. coli cells. According to the protocol, ligation and preparation of competent cells is performed in parallel. Therefore, it takes only one hour from the completion of PCR to cell plating. Our transformation protocol is often faster than the transformation of commercially available competent cells. The DNA insert can be readily excised from the versatile polylinker of pTZ57R/T, sequenced using standard M13/pUC primers or in vitro transcribed with T7 RNA polymerase.

Quality Control

The kit is functionally tested by the cloning efficiency of a control PCR product. Typical yield of the recombinant clones is higher than 90%. The transformation system - TransformAid™ Bacterial Transformation Kit is tested in transformation of E.coli strains XL1-Blue and JM107 with pUC19 DNA. Typical transformation efficiency is more than 10⁷ transformants per µg of supercoiled pUC19 DNA.

Components

Vector pTZ57R/T (pTZ57R DNA sequence in txt format)
T4 DNA Ligase
5X Ligation Buffer
Control PCR Fragment (sequence of control PCR product in txt format)
Control DNA 1
Control DNA 2
Water, nuclease-free
TransformAid™ Bacterial Transformation System:
- C-Medium;
- T-Solution (A);
- T-Solution (B)
Detailed Protocol

Note

E.coli strains are not included.
Suitable for all common laboratory E.coli strains.

Figure 1. Restriction map of vector pTZ57R/T.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

References

Clark, J.M., Novel non-templated nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerases, Nucl. Acids Res., 16 (20), 9677-9686, 1988.

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