N-His-pTrham Fast Cloning Kit | Toxic Protein Expression

Amid Biosciences pTrham Fast Cloning Kits facilitate rapid cloning and gene expression in E. coli. The N-His-pTrham Cloning Kit (catalog # NHPTR-401) consists of linearized N-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.

N-His-pTrham Fast Cloning Kit Components (5 reactions) 

• Linearized N-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 

The N-His-pTrham vector, together with C-His-pTrham, represent a unique pair of expression vectors having the same plasmid backbone for production of recombinant proteins fused only to a 6X histidine tag sequence at the N- or C-terminus. Linearized N-His-pTrham and C-His-pTrham are based on Amid Biosciences pTrham vector (catalog # PTR-401) and have same features as pTrham designed for providing the highest levels of expression of recombinant proteins: the rhaP_BAD promoter, the rrnG antitermination region, the bacteriophage T7 gene 10 translation enhancer and ribosome binding site. The vectors contain an ampicillin resistance marker. L-rhamnose-inducible rhaP_BAD 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.

Benefits:
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
Store all kit components except competent cells at -20 ^oC. Competent cells must be stored at -80 ^oC.

International Shipping:  Product requires shipping on ice packs. Please contact info@amidbiosciences.com for shipment estimates. 

This product is intended for research use only.

Rhamnose Expression and Cloning System - Manual

Important Note: Purchase of the N-His-pTrham vector includes a license for use only at site of purchase and may not be distributed to other sites.

License: The N-His-pTrham vector when purchased conveys a limited use license to the end user. This license prohibits the purchaser from selling, assigning or transferring this product to any third party without the express written consent of Amid Biosciences, LLC. Please review this license before purchasing this product.