Background: Plasmid building of small fragments of curiosity (similar to insertion of small fragment marker genes, expression of shRNA, siRNA, and many others) is the idea of many biomolecular experiments. Right here, we describe a technique to clone quick DNA into vectors by polymerase chain response (PCR), named one-step PCR cloning. Our methodology makes use of PCR to amplify your complete round plasmid. The PCR was carried out by the primers containing the gene of quick DNA with overlapping sequences between 10-15 bp. The PCR merchandise had been then remodeled into E. coli and cyclized by homologous recombination in vivo.
Strategies: The pEGFP-N1-HA plasmid was constructed by one-step PCR and transformation. Cells had been transfected with pEGFP-N1-HA and pEGFP-N1 plasmid utilizing TurboFect transfection reagent. Protein expression was detected by western blotting and the HA-GFP fusion protein was detected by confocal microscopy.
Outcomes: The pEGFP-N1-HA plasmid was efficiently constructed and HA expression in cells.
Conclusions: Free from the restrictions of restriction enzyme websites and omitting the ligation course of, our methodology provides a versatile and economical possibility of plasmid building.
Key factors: Vital findings of the research A technique to clone quick DNA into plasmids was discovered. What this research provides Our research supplies a versatile and economical choice to clone quick DNA into plasmids.
Selective isolation of huge segments from particular person microbial genomes and environmental DNA samples utilizing transformation-associated recombination cloning in yeast.
Right here, we describe an extension of our unique transformation-associated recombination (TAR) cloning protocol, enabling selective isolation of DNA segments from microbial genomes. The approach is predicated on the beforehand described TAR cloning process developed for isolation of a fascinating area from mammalian genomes which are enriched in autonomously replicating sequence (ARS)-like sequences, components that operate because the origin of replication in yeast. Such sequences aren’t widespread in microbial genomes. On this Protocol Extension, an ARS is inserted into the TAR vector together with a counter-selectable marker, permitting for choice of cloning occasions towards vector circularization.
Pre-treatment of microbial DNA with CRISPR-Cas9 to generate double-stranded breaks close to the focused sequences tremendously will increase the yield of region-positive colonies. Compared to different accessible strategies, this Protocol Extension permits selective isolation of any area from microbial genomes in addition to from environmental DNA samples. Your complete process could be accomplished in 10 d. Generally used and nicely accepted approaches are missing for site-directed modification of adenoviral vectors. Right here, we try to introduce an easy-to-implement technique for such objective with an instance of creating a replication competent adenoviral vector system from pKAd5 plasmid, an infectious clone of human adenovirus 5 (HAdV-5). PCR merchandise of GFP expression cassette and plasmid spine had been fused with the EcoRI/NdeI-digested fragment of pKAd5 to generate a modified intermediate plasmid pMDXE3GA by DNA meeting.
NdeI-digested fragment of pMDXE3GA was introduced again to pKAd5 to kind the adenoviral plasmid pKAd5XE3GA by restriction-ligation cloning. Recombinant adenovirus HAdV5-XE3GA was rescued, amplified and purified. The expression of GFP and the propagation of virus in adherent HEp-2 and suspension Ok562 cells had been investigated. Expression of goal gene was considerably enhanced in each cell traces contaminated with HAdV5-XE3GA as a result of virus replication.
Cloning short DNA into plasmids by one-step PCR
Common Template-Assisted, Cloning-free Methodology for the Technology of Small RNA-Expressing Dumbbell-Formed DNA Vectors.
Dumbbell-shaped DNA minimal vectors symbolize genetic vectors solely composed of the gene expression cassette of curiosity and terminal closing loop buildings. Dumbbell vectors for small hairpin RNA or microRNA expression are extraordinarily small-sized, which is advantageous with regard to mobile supply and nuclear diffusion. Standard methods for the era of small RNA-expressing dumbbell vectors require cloning of a respective plasmid vector, which is subsequently used for dumbbell manufacturing. Right here, we current a novel cloning-free methodology for the era of small RNA-expressing dumbbell vectors that additionally doesn’t require any restriction endonucleases.
This new PCR-based methodology makes use of a common DNA template comprising an inverted repeat of the minimal H1 promoter and the miR-30 stem. The sequences coding for small RNA expression are launched by the PCR primers. Dumbbells are shaped by denaturing and reannealing of the PCR product and are covalently closed utilizing ssDNA ligase. The brand new protocol generates plus- and/or minus-strand dumbbells, each of which had been proven to set off environment friendly goal gene knockdown.
) Cytokeratin, Pan; Clones AE1 & AE3 (Concentrate) |
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| A00152-C.1 | ScyTek Laboratories | 0.1 ml | EUR 138 |
) Chromogranin A; Clones LK2H10 & PHE5 (Concentrate) |
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| A00160-C | ScyTek Laboratories | 1 ml | EUR 488 |
Chromogranin A; Clones LK2H10 & PHE5 (Concentrate) |
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| A00160-C.1 | ScyTek Laboratories | 0.1 ml | EUR 136 |
 Multiple Endocrine Neoplasia I Antibody |
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| 20-abx113892 | Abbexa |
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 Multiple Line Reader |
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| LFRB-001 | BioAssayWorks | Each | EUR 3916.66 |
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Description: Multiple Line Reader is a portable universal lateral-flow reader that is based on colour change method. |
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-Like 3 (EXTL3) Antibody) Exostoses (Multiple)-Like 3 (EXTL3) Antibody |
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| 20-abx002804 | Abbexa |
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Exostoses (Multiple)-Like 3 (EXTL3) Antibody |
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| 20-abx212396 | Abbexa |
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Exostoses (Multiple)-Like 3 (EXTL3) Antibody |
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| 20-abx212881 | Abbexa |
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-Like 2 (EXTL2) Antibody) Exostoses (Multiple)-Like 2 (EXTL2) Antibody |
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| 20-abx112417 | Abbexa |
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Exostoses (Multiple)-Like 3 (EXTL3) Antibody |
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| 20-abx112418 | Abbexa |
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Exostoses (Multiple)-Like 3 (EXTL3) Antibody |
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| abx036241-100ug | Abbexa | 100 ug | EUR 391 |
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Exostoses (Multiple)-Like 2 (EXTL2) Antibody |
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| abx036893-100ug | Abbexa | 100 ug | EUR 391 |
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 Antibody) Multiple Endocrine Neoplasia I (MEN1) Antibody |
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| 20-abx128411 | Abbexa |
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Exostoses (Multiple)-Like 3 (EXTL3) Antibody |
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| 20-abx109769 | Abbexa |
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-Like 1 (EXTL1) Antibody) Exostoses (Multiple)-Like 1 (EXTL1) Antibody |
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| abx030322-400ul | Abbexa | 400 ul | EUR 523 |
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Exostoses (Multiple)-Like 1 (EXTL1) Antibody |
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| abx030322-80l | Abbexa | 80 µl | EUR 286 |
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Exostoses (Multiple)-Like 3 (EXTL3) Antibody |
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| abx034206-400ul | Abbexa | 400 ul | EUR 523 |
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Exostoses (Multiple)-Like 3 (EXTL3) Antibody |
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| abx034206-80l | Abbexa | 80 µl | EUR 286 |
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Exostoses (Multiple)-Like 2 (EXTL2) Antibody |
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| abx034288-400ul | Abbexa | 400 ul | EUR 523 |
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Exostoses (Multiple)-Like 2 (EXTL2) Antibody |
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| abx034288-80l | Abbexa | 80 µl | EUR 286 |
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Exostoses (Multiple)-Like 2 (EXTL2) Antibody |
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| abx232911-100ug | Abbexa | 100 ug | EUR 509 |
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Exostoses (Multiple)-Like 3 (EXTL3) Antibody |
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| abx232912-100ug | Abbexa | 100 ug | EUR 481 |
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 Leukemia - Multiple Myeloma Exosome |
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| P141-MM | 101Bio | NULL | EUR 0 |
 Recombinant HCV Multiple Antigen |
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| VAng-Lsx0198-1mg | Creative Biolabs | 1 mg | EUR 2800 |
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Description: HCV Multiple Antigen, recombinant protein from E. coli. |
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) Anti-HPV 6 L1 monoclonal antibody (6 clones per set) |
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| CABT-B8803 | Creative Diagnostics | 20 ug× 6 clones | EUR 1677 |
) Anti-HPV 11 L1 monoclonal antibody (6 clones per set) |
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| CABT-B8804 | Creative Diagnostics | 20 ug× 6 clones | EUR 1677 |
) Anti-HPV 16 L1 monoclonal antibody (6 clones per set) |
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| CABT-B8805 | Creative Diagnostics | 20 ug× 6 clones | EUR 1677 |
) Anti-HPV 18 L1 monoclonal antibody (6 clones per set) |
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| CABT-B8806 | Creative Diagnostics | 20 ug× 6 clones | EUR 1677 |
) Anti-HPV 31 L1 monoclonal antibody (6 clones per set) |
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| CABT-B8807 | Creative Diagnostics | 20 ug× 6 clones | EUR 1677 |
) Anti-HPV 33 L1 monoclonal antibody (6 clones per set) |
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| CABT-B8808 | Creative Diagnostics | 20 ug× 6 clones | EUR 1677 |
) Anti-HPV 45 L1 monoclonal antibody (6 clones per set) |
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| CABT-B8809 | Creative Diagnostics | 20 ug× 6 clones | EUR 1677 |
) Anti-HPV 52 L1 monoclonal antibody (6 clones per set) |
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| CABT-B8810 | Creative Diagnostics | 20 ug× 6 clones | EUR 1677 |
) Anti-HPV 58 L1 monoclonal antibody (6 clones per set) |
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| CABT-B8811 | Creative Diagnostics | 20 ug× 6 clones | EUR 1677 |
 p53 Cocktail; Clones Pab 1801 plus DO-7 |
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| A00093 | ScyTek Laboratories | 6 ml | EUR 282 |
p53 Cocktail; Clones Pab 1801 plus DO-7 |
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| A00093.0025 | ScyTek Laboratories | 25 ml | EUR 725 |
) Napsin A; Clones NAPSA/1238 & NAPSA/1239 (Concentrate) |
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| A00131-C | ScyTek Laboratories | 1 ml | EUR 488 |
Napsin A; Clones NAPSA/1238 & NAPSA/1239 (Concentrate) |
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| A00131-C.1 | ScyTek Laboratories | 0.1 ml | EUR 136 |
) MART-1; Clones M2-7C10 & M2-9E3 (Concentrate) |
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| A00133-C | ScyTek Laboratories | 1 ml | EUR 484 |
) Cytokeratin, Pan; Clones AE1 & AE3 (Ready-To-Use) |
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| A00152-0002 | ScyTek Laboratories | 2 ml | EUR 104 |
Cytokeratin, Pan; Clones AE1 & AE3 (Ready-To-Use) |
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| A00152-0007 | ScyTek Laboratories | 7 ml | EUR 183 |
Cytokeratin, Pan; Clones AE1 & AE3 (Ready-To-Use) |
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| A00152-0025 | ScyTek Laboratories | 25 ml | EUR 449 |
) Chromogranin A; Clones LK2H10 & PHE5 (Ready-To-Use) |
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| A00160-0002 | ScyTek Laboratories | 2 ml | EUR 101 |
Chromogranin A; Clones LK2H10 & PHE5 (Ready-To-Use) |
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| A00160-0007 | ScyTek Laboratories | 7 ml | EUR 178 |
Chromogranin A; Clones LK2H10 & PHE5 (Ready-To-Use) |
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| A00160-0025 | ScyTek Laboratories | 25 ml | EUR 435 |
p53 Cocktail; Clones Pab 1801 plus DO-7 |
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| A20093 | ScyTek Laboratories | 2 ml | EUR 165 |
 Antibody) Multiple EGF Like Domains 9 (MEGF9) Antibody |
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| 20-abx007525 | Abbexa |
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Multiple EGF Like Domains 9 (MEGF9) Antibody |
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| abx027366-400ul | Abbexa | 400 ul | EUR 523 |
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Multiple EGF Like Domains 9 (MEGF9) Antibody |
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| abx027366-80l | Abbexa | 80 µl | EUR 286 |
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 Antibody) Multiple Inositol Polyphosphate Phosphatase 1 (MINPP1) Antibody |
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| abx028250-400ul | Abbexa | 400 ul | EUR 523 |
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Multiple Inositol Polyphosphate Phosphatase 1 (MINPP1) Antibody |
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| abx028250-80l | Abbexa | 80 µl | EUR 286 |
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 Antibody) EGF Like Domain Multiple 8 (EGFL8) Antibody |
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| 20-abx213757 | Abbexa |
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 Antibody) Multiple EGF Like Domains 8 (MEGF8) Antibody |
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| abx215087-100ug | Abbexa | 100 ug | EUR 439 |
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-Like 3 (EXTL3) Antibody (Biotin)) Exostoses (Multiple)-Like 3 (EXTL3) Antibody (Biotin) |
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| 20-abx105397 | Abbexa |
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-Like 3 (EXTL3) Antibody (FITC)) Exostoses (Multiple)-Like 3 (EXTL3) Antibody (FITC) |
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| 20-abx106816 | Abbexa |
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-Like 3 (EXTL3) Antibody (HRP)) Exostoses (Multiple)-Like 3 (EXTL3) Antibody (HRP) |
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| 20-abx108235 | Abbexa |
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 Rna Binding Protein With Multiple Splicing Antibody |
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| 20-abx115330 | Abbexa |
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-Like 3 (EXTL3) Antibody Pair) Exostoses (Multiple)-Like 3 (EXTL3) Antibody Pair |
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| abx117473-1pair5x96wellplates | Abbexa | 1 pair (5x96 well plates) | EUR 1010 |
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Multiple Inositol Polyphosphate Phosphatase 1 (MINPP1) Antibody |
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| 20-abx126159 | Abbexa |
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 Antibody) EGF Like Domain Multiple 6 (EGFL6) Antibody |
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| abx036838-100ug | Abbexa | 100 ug | EUR 391 |
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Multiple Inositol Polyphosphate Phosphatase 1 (MINPP1) Antibody |
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| abx038060-100ug | Abbexa | 100 ug | EUR 391 |
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Multiple Inositol Polyphosphate Phosphatase 1 (MINPP1) Antibody |
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| 20-abx133721 | Abbexa |
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Multiple Inositol Polyphosphate Phosphatase 1 (MINPP1) Antibody |
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| 20-abx109332 | Abbexa |
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EGF Like Domain Multiple 8 (EGFL8) Antibody |
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| abx122807-100ug | Abbexa | 100 ug | EUR 391 |
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 Antibody) EGF Like Domain Multiple 7 (EGFL7) Antibody |
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| abx145646-100ug | Abbexa | 100 ug | EUR 391 |
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Multiple EGF Like Domains 9 (MEGF9) Antibody |
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| 20-abx014606 | Abbexa |
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Multiple Inositol Polyphosphate Phosphatase 1 (MINPP1) Antibody |
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| 20-abx014607 | Abbexa |
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EGF Like Domain Multiple 8 (EGFL8) Antibody |
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| 20-abx241508 | Abbexa |
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 Antibody) Multiple Inositol Polyphosphate Phosphatase 1 (Minpp1) Antibody |
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| 20-abx319662 | Abbexa |
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Multiple Inositol Polyphosphate Phosphatase 1 (Minpp1) Antibody |
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| 20-abx319666 | Abbexa |
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 Antibody) Multiple inositol polyphosphate phosphatase 1 (MINPP1) Antibody |
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| abx331046-100ul | Abbexa | 100 ul | EUR 425 |
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Multiple EGF Like Domains 9 (MEGF9) Antibody |
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| abx331804-100ul | Abbexa | 100 ul | EUR 425 |
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Multiple EGF Like Domains 9 (MEGF9) Antibody |
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| 20-abx327823 | Abbexa |
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Multiple inositol polyphosphate phosphatase 1 (MINPP1) Antibody |
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| 20-abx328388 | Abbexa |
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Multiple EGF Like Domains 8 (MEGF8) Antibody |
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| 20-abx324529 | Abbexa |
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Multiple EGF Like Domains 9 (MEGF9) Antibody |
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| 20-abx338519 | Abbexa |
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EGF Like Domain Multiple 7 (EGFL7) Antibody |
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| abx432640-200ul | Abbexa | 200 ul | EUR 384 |
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EGF Like Domain Multiple 6 (EGFL6) Antibody |
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| 20-abx302793 | Abbexa |
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EGF Like Domain Multiple 7 (EGFL7) Antibody |
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| abx232666-100ug | Abbexa | 100 ug | EUR 481 |
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 polyclonal antibody) Multiple endocrine neoplasia 1 (Men1) polyclonal antibody |
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| ABP-PAB-10831 | Allele Biotech | 100 ug | Ask for price |
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 polyclonal antibody) Multiple endocrine neoplasia 1 (MEN1) polyclonal antibody |
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| ABP-PAB-10832 | Allele Biotech | 100 ug | Ask for price |
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 MAP kinase fragment [Multiple species] |
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| A1079-1 | ApexBio | 1 mg | EUR 108 |
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Description: This Mitogen-activated protein kinase (MAP kinase) fragment has a peptide sequence of Lys-Tyr-Ile-His-Ser-Ala-Asn-Val-Leu.The MAP kinases are serine/threonine-specific protein kinasesbelonging to the CMGC (CDK/MAPK/GSK3/CLK) kinase group. |
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MAP kinase fragment [Multiple species] |
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| A1079-10 | ApexBio | 10 mg | EUR 340 |
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Description: This Mitogen-activated protein kinase (MAP kinase) fragment has a peptide sequence of Lys-Tyr-Ile-His-Ser-Ala-Asn-Val-Leu.The MAP kinases are serine/threonine-specific protein kinasesbelonging to the CMGC (CDK/MAPK/GSK3/CLK) kinase group. |
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MAP kinase fragment [Multiple species] |
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| A1079-25 | ApexBio | 25 mg | EUR 456 |
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Description: This Mitogen-activated protein kinase (MAP kinase) fragment has a peptide sequence of Lys-Tyr-Ile-His-Ser-Ala-Asn-Val-Leu.The MAP kinases are serine/threonine-specific protein kinasesbelonging to the CMGC (CDK/MAPK/GSK3/CLK) kinase group. |
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MAP kinase fragment [Multiple species] |
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| A1079-5 | ApexBio | 5 mg | EUR 224 |
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Description: This Mitogen-activated protein kinase (MAP kinase) fragment has a peptide sequence of Lys-Tyr-Ile-His-Ser-Ala-Asn-Val-Leu.The MAP kinases are serine/threonine-specific protein kinasesbelonging to the CMGC (CDK/MAPK/GSK3/CLK) kinase group. |
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 Leukemia - Multiple Myeloma Exosome RNA |
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| P241-MM | 101Bio | NULL | EUR 0 |
) Cytokeratin 5,6 Cocktail; Clones EP42 & EP67 (Ready-To-Use) |
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| A00141-0002 | ScyTek Laboratories | 2 ml | EUR 268 |
Cytokeratin 5,6 Cocktail; Clones EP42 & EP67 (Ready-To-Use) |
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| A00141-0007 | ScyTek Laboratories | 7 ml | EUR 594 |
Cytokeratin 5,6 Cocktail; Clones EP42 & EP67 (Ready-To-Use) |
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| A00141-0025 | ScyTek Laboratories | 25 ml | EUR 1683 |
 Antibody) Multiple Coagulation Factor Deficiency Protein 2 (MCFD2) Antibody |
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| abx025215-400ul | Abbexa | 400 ul | EUR 523 |
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Multiple Coagulation Factor Deficiency Protein 2 (MCFD2) Antibody |
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| abx025215-80l | Abbexa | 80 µl | EUR 286 |
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 Antibody) RNA-Binding Protein With Multiple Splicing (RBPMS) Antibody |
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| 20-abx003350 | Abbexa |
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 Antibody) EGF Like Domain Protein, Multiple 6 (EGFL6) Antibody |
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| 20-abx176223 | Abbexa |
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 Antibody) EGF Like Domain Protein, Multiple 7 (EGFL7) Antibody |
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| 20-abx176224 | Abbexa |
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EGF Like Domain Protein, Multiple 7 (EGFL7) Antibody |
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| 20-abx212160 | Abbexa |
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 Antibody) CUB And Sushi Multiple Domains 1 (CSMD1) Antibody |
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| 20-abx212966 | Abbexa |
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CUB And Sushi Multiple Domains 1 (CSMD1) Antibody |
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| 20-abx212967 | Abbexa |
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 Antibody (Biotin)) Multiple Inositol Polyphosphate Phosphatase 1 (MINPP1) Antibody (Biotin) |
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| 20-abx105681 | Abbexa |
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 Antibody (FITC)) Multiple Inositol Polyphosphate Phosphatase 1 (MINPP1) Antibody (FITC) |
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| 20-abx107098 | Abbexa |
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 Antibody (HRP)) Multiple inositol polyphosphate phosphatase 1 (MINPP1) Antibody (HRP) |
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| 20-abx108519 | Abbexa |
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This methodology allows quick, low cost manufacturing of small RNA-expressing dumbbell vectors in a excessive throughput-compatible method for practical genomics screens or, as dumbbells aren’t liable to transgene silencing, for knockdown research in main cells.