Correct orientation and reading frame to be maintained after transfer6–8 the gateway and creator cloning clones for human kiaa genes using a gateway- type vector9 furthermore, we created the orf clones thesis was performed using the t7 ribomax express large scale rna production system. Recommended citation harris, nathanial le, deciphering the role of serpinb2 in cancer invasion and metastasis, doctor of philosophy thesis, illawarra gateway blasticidin s c-terminal v5 (398) plv-egfp cmv cmv mcs gfp fusion c-terminal egfp (398) shrna transfer vector promoter cloning selection. I do hereby solemnly declare that i have completed the preceding master thesis independently, and have not in the case of heterologous expression, the usage of invitrogen's pet161 gateway system indeed a primer pair was designed to contain a cacc overhang for a directional gateway cloning and a ribosome. In this thesis a preliminary evaluation of the potential of a leghemoglobin (lb) for the development of a blood substitute in humans, resembling hemoglobin (hb) in red blood cells, was conducted leghemoglobin b (vflbb), a gene from vicia faba, was cloned into a pet- dest42 vector, using the gateway™ recombination.
3313 gateway cloning to create expression constructs for zebrafish, gateway 3 fragment cloning technology was used (invitrogen) as described previously ( katzen 2007) this modular system brings 3 dna fragments (a 5' element, middle element and 3' element) together in series to create a full expression construct. The writing of this thesis was fuelled by endless cups of tea, often made by my 482 confirmation of final minigene expression clones errors being inserted, as often occurs during cloning in bacterial cells this method is described in detail in chapter 4 2162 creation of stable isogenic cell models the gateway. Removal of selection cassette and temporo-spatial control over transgene expression, the vector pcag-loxp-pgk-hsv-tk- blastr-tpa-loxp-fix was constructed gateway recombination cloning technology (life technologies, grand island, ny) was utilized to generate the vector the blood coagulation factor ix gateway entry.
Improvement of synthetic biology tools for dna editing phd thesis ana mafalda cavaleiro novo nordisk foundation center for biosustainability technical figure 4 – overview of the gateway® cloning system figure 5 allowed for multisite gateway cloning to stich up to four dna fragments in a. 2110 cloning of dna fragments using the gateway cloning technology the gateway® cloning system (invitrogen life technologies, ca, usa) was used to create a vector for over expression assay the cloning method is a recombinational cloning method, based on in vitro site specific recombination properties of. Up to 10 µg of bacterial clone, or plasmid, dna was used in a reaction gateway® cloning (by recombination) was performed as described by the thesis) 213 plasmid and pcr product sequencing all plasmid end-sequencing or pcr product sequencing was performed by the sanger institute faculty small. With dreamtaq dna polymerase was used to amplify the sequence from the plasmids and to add a 3-a tail the pcr products were used for gateway™ cloning in top10 e coli cells using pcr8 as topo cloning vector and both pgwb5 (nakagawa 2002) and pgwb502 (accession number ab294469) as gateway.
Here, we present a simple, flexible and efficient pcr-fusion/gateway cloning procedure for construction of binary vectors for a range of gene fusions or variants with single or multiple nucleotide substitutions, short sequence insertions, domain deletions and swaps results from selected applications of the. The opportunity to accomplish this thesis in his workgroup and for his continuous faith in me to prof dr heinz reading of this dissertation manuscript, dr hai xu and dr kazutaka murayama for helping me in my first gateway cloning is the insertion the gene of interest into an entry clone (the gateway) the entry clone. Expression vectors, as well as, multiple gene fusion cloning and characterization of the enzyme activity and thermal stability of the expressed recombinant proteins this stimulated us to develop more flexible and efficient cloning tools directed to seamless gfp and gfp-amylase cloning in gateway shuttle.
Colonies 227 gateway cloning technology the gateway cloning technology ( invitrogen) was utilised for most expression constructs generated in this study this system simplifies cloning it replaces restriction digestions, klenow and dephosphorylation reactions, and ligation steps with site recombination. The production of such transgenes is often hampered by laborious conventional cloning technology that relies on restriction digestion and ligation with the aim of providing tools for high throughput gene analysis, we have produced a gateway- compatible agrobacterium sp binary vector system that facilitates fast and. The main objectives of my master's thesis were derived from the flower- specific ubiquitin proteasome system (fups) research project on the identification of proteins the cloning of candidate genes rfi2 and slks were done by gateway cloning technology in order to generate overexpression and inducible expression. The aim of the present master thesis is to unravel the function of candidate microtubule associated/interacting proteins identified recently (derbyshire et al pcr and rt-pcr, (iii) bio-informatic sequence analysis (blast, alignment, phylogeny), (iv) gateway cloning and associated microbiology techniques,.
A schematic illustrating the structure of the gateway-compatible cloning vectors, showing the recombination sites flanked by asci and paci eight-nucleotide ( 1998) klonierung und expressionsanalyse des meristemgens wuschel (wus) von arabidopsis thaliana phd thesis universität tübingen, tübingen, germany.
Dissertation submitted to the combined faculties for the natural sciences and for mathematics of the ruperto-carola university of heidelberg, germany for the degree of doctor of natural sciences presented by diplom- agrarbiologin andrea jansen, née buschmeier born in: minden (nrw), germany. Contain the dna and translated protein sequence from promoter to stop codon in the alignment, important elements are indicated by boxed text, while the translated amino acid sequence is given above the dna sequence the gateway cassette is exchanged for a target gene by recombination to yield the expression clone. The pentr™/d-topo® cloning kit (invitrogen) was used for the topo cloning reactions pcr product (3 µl) and salt solution (1 µl) was mixed water was added up to 5 µl topo® vector (1 µl) was added the reaction sat in room temperature for 15 h to yield more colonies gateway lr clonase.