C-His-pTrham Fast Cloning Kit | Amid Biosciences
Amid Biosciences pTrham Fast Cloning Kits facilitate rapid cloning and gene expression in E. coli. The C-His-pTrham Cloning Kit (catalog # CHPTR-401) consists of linearized C-His-pTrham Vector that allows cloning of any PCR product, E. coli AB 5-alpha Chemically Competent Cells for cloning and protein expression, control insert DNA, and sequencing primers for clone analysis. Cloning into C-His-pTrham vector results to production of recombinant proteins fused only to a 6X histidine tag sequence at the C-terminus.
C-His-pTrham Fast Cloning Kit Components (5 reactions)
- Linearized C-His-pTrham (20 ng/µl): 10 µl
- Positive control: a PCR amplicon of GFP, 753 bp (20 ng/µl): 5 µl
- Sequencing Primers:
- Forward pTrham (5 µM) - 10 µl
- Reverse pTrham (5 µM) - 10 µl
- E. coli AB 5-alpha chemically competent cells : 5 X 100 µl
Linearized C-His-pTrham vector is based on Amid Biosciences pTrham vector (catalog # PTR-401) and has same features as pTrham designed for providing the highest levels of expression of recombinant proteins: the rhaPBAD promoter, the rrnG antitermination region, the bacteriophage T7 gene 10 translation enhancer and ribosome binding site. The vector contains an ampicillin resistance marker. L-rhamnose-inducible rhaPBAD promoter is capable of high expression level and ensures minimal basal expression in the absence of inducer. This level of control is particularly useful for expression of genes encoding toxic products. pTrham vector series can be used in any E. coli strains.
The cloning strategy is based on in vivo recombination of the linear vector and insert DNA fragment containing common flanking sequences. The recombination-mediated approach enables directional cloning of any PCR products into the vector without the need for restriction enzyme digestion or ligation reactions. Inserts designed for cloning into pTrham linearized vectors are generated by PCR with primers having 15 to 20 bases of homology with the ends of the vector. The PCR product is mixed with the corresponding cloning-ready vector and transformed into competent E. coli cells. No further enzymatic step after PCR amplification or purification of the vector and inserts are required. At least two PCR fragments can be assembled simultaneously into a vector with a high efficiency. Compared with the conventional restriction-ligation-dependent or other ligase-independent cloning approaches, the recombinational cloning is simplest and fastest method available. In addition to simplicity, the system is very robust and highly efficient.
- Universal - clone any insert
- Easy – simply mix and transform
- Fast – no further enzymatic step after PCR amplification
- Seamless - no extra nucleotides
- Efficient > 95% positive colonies
- Economical – saving on time and reagents
Storage: -20 °C
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This product is intended for research use only.