Gateway® Vector Construction and Cloning Services

By: Genesys Design  09-12-2011

We also provide a high-throughput cloning service that follows the protocol outlined below.

  1. Preparation. We review the nucleotide sequence(s) that you provide to confirm the complete ORF sequence. Primers will be designed to flank the initiating methionine (ATG) and the stop codon. In cases where C-terminal fusions are desired, the open reading frames may be amplified without any stop codon (Figure 1).
  2. Amplification. Low-cycle, high-fidelity amplification of the desired ORF from a plasmid template.
  3. Screening. We will screen Invitrogen’s collection of full-length cDNA libraries if no appropriate plasmid template is available.
  4. Cloning. Our scientists will clone the amplified ORF into a Gateway® Entry vector and transform the DNA into chemically competent E. coli (Figure 2).
  5. Sequencing. 5' and 3' end–read sequencing in order to verify amplification of the correct target as well as correct reading frame.
  6. Verification. Screening of antibiotic-resistant colonies to confirm positive Entry clones.
  7. Delivery. You receive the confirmed Entry clone(s) as glycerol stocks. You may have the Entry clone(s) transferred to one or more Gateway® Destination vectors to create expression clones suitable for immediate application in the corresponding expression system(s).

Figure 2. High-Throughput ORF Cloning: 

Representative full-length gene expressed within an Invitrogen™ full-length cDNA library. Since the gene has been cloned into the pCMV•Sport 6.1 vector, it is flanked by the attB1 and attB2 Gateway® recombination sites. The gene is composed of an open reading frame, illustrated as the area between the initiating methionine (ATG) and the stop codon. The open reading frame is that portion of the gene which codes for a functional protein. Flanking the open reading frame are 5´ and 3´ untranslated regions, which contain motifs involved in, for example, regulation, transcription, and posttranslational modifications.

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