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ORF Cloning Services
Open reading frame (ORF) cloning provides the means to turn DNA sequences into expression-ready clones. Open reading frames from fulllength genes are amplified and cloned into Gateway® entry vectors. The resulting high-fidelity cloned ORFs provide:
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Access to Gateway® Technology
Through our high-throughput ORF cloning service, Invitrogen provides you access to Gateway® Technology, a universal system for protein expression and analysis based on lambda-phage recombination. Gateway® Technology enables the transfer of DNA among different vectors without time-consuming sub-cloning steps. Once a gene is converted into a Gateway® entry clone, it is easily moved into one or more vectors in the desired orientation and reading frame. Supply a full-length cDNA sequence, and our scientists will amplify the open reading frame and subsequently clone the PCR product into a Gateway® entry vector. You can then transfer the ORF into any Gateway® expression vector. ORF cloning is particularly valuable if you are conducting gene functional analysis, where the 5´ and 3´ untranslated regions of fulllength genes can interfere with optimal protein expression.
High-Throughput ORF Cloning Service
Figure 1. 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 is 5´ and 3´ untranslated regions, which contain motifs involved in, for example, regulation, transcription and post-translational modifications.
Figure 2. PCR-based ORF cloning
ORFs are amplified using gene-specific primers which contain attB1 and attB2 recombination sites. The resulting amplicon may be recombined with an attP donor vector (pDONR™) in a B x P reaction to yield an entry clone. ORFs contained within entry clones are flanked by attL sites and may be subsequently recombined with any Gateway® destination vector to yield expression clones which may, in return, be used in functional studies.
Through our high-throughput ORF cloning service, Invitrogen provides you access to Gateway® Technology, a universal system for protein expression and analysis based on lambda-phage recombination. Gateway® Technology enables the transfer of DNA among different vectors without time-consuming sub-cloning steps. Once a gene is converted into a Gateway® entry clone, it is easily moved into one or more vectors in the desired orientation and reading frame. Supply a full-length cDNA sequence, and our scientists will amplify the open reading frame and subsequently clone the PCR product into a Gateway® entry vector. You can then transfer the ORF into any Gateway® expression vector. ORF cloning is particularly valuable if you are conducting gene functional analysis, where the 5´ and 3´ untranslated regions of fulllength genes can interfere with optimal protein expression.
High-Throughput ORF Cloning Service
- 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).
- Amplification. Low-cycle, high-fidelity amplification of the desired ORF from a plasmid template.
- Screening. We will screen Invitrogen’s collection of full-length cDNA libraries if no appropriate plasmid template is available.
- Cloning. Our scientists will clone the amplified open reading frame into a Gateway® entry vector and transform the DNA into chemically competent E. coli (Figure 2).
- Sequencing. 5' and 3' end-read sequencing in order to verify amplification of the correct target as well as correct reading frame.
- Verification. Screening of antibiotic-resistant colonies to confirm positive entry clones.
- 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 1. 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 is 5´ and 3´ untranslated regions, which contain motifs involved in, for example, regulation, transcription and post-translational modifications.
Figure 2. PCR-based ORF cloning
ORFs are amplified using gene-specific primers which contain attB1 and attB2 recombination sites. The resulting amplicon may be recombined with an attP donor vector (pDONR™) in a B x P reaction to yield an entry clone. ORFs contained within entry clones are flanked by attL sites and may be subsequently recombined with any Gateway® destination vector to yield expression clones which may, in return, be used in functional studies.
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