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Megawhop pcr

Web3 jun. 2016 · The purified PCR products were used as primers for MEGAWHOP PCR. The reactions were carried out in 50 μl containing 3 μl Template DNA pET-15b-ESH, 10 μl mega primers (the purified error-prone PCR products), 1 μl dNTP Mix, 5 μl Taq plus buffer, 1 μl Taq plus polymerase, 1 μl Pfu polymerase and 29 μl deionized water. WebMEGAWHOP PCR: 2×High Fidelity Master Mix DpnI digestion: DpnI (NEB) (20,000units/ml) 10xCutsmart Product purification E.Z.N.A.® Cycle Pure Kit Please wear gloves for the …

Rapid Sequence Scanning Mutagenesis Using - Springer

Web1 jan. 2011 · MEGAWHOP allows for the cloning of DNA fragments into a vector and is used for conventional restriction digestion/ligation-based procedures. In MEGAWHOP, the DNA fragment to be cloned is used as a set of complementary primers that replace a homologous region in a template vector through whole-plasmid PCR. Web50 µL. The MEGAWHOP PCR (68°C for 5 min, 1 cycle; 95°C for 2 min, 1 cycle; 95°C for 30 s/55°C for 30 s/68°C for 12 min, 18 cycles; 68°C for 30 min, 1 cycle) was performed in a final volume of 50 µL using dNTP mix (0.2 mM), megaprimer (puri-fied epPCR amplification product, 400 ng), plasmid DNA tem-plate (pBCSSC, 50 ng) and . Pfu toddlers trampoline with a bar https://jdgolf.net

Improvement of a newly cloned carbonyl reductase and its …

Web16 nov. 2024 · Wang et al. describe a new chemo-enzymatic strategy toward the diversity-orientated synthesis of enantiopure cyclohexenes. This strategy features an intermolecular Diels-Alder reaction catalyzed by an engineered Diels-Alderase followed by various transition-metal-catalyzed decarboxylative functionalizations. Both stereoselectivity and … Web7 apr. 2024 · The PCR product containing all four mutagenesis sites (79, 161, 246 and 296) was used as megaprimer to perform MEGAWHOP PCR 44. DpnI digestion (20 U) of the template was performed at 37 °C for... Web1 feb. 2003 · MEGAWHOP was carried out in Taq PCR buffer, 50 ng of pHYHP200 plasmid template, 500 ng of SeSaM library PCR product from step 4, 0.3 mM of each dNTPs and … toddlers \u0026 tiaras season 9 episode 13

Directed evolution of the PobR allosteric transcription factor to ...

Category:Construction of mutant library - GitHub Pages

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Megawhop pcr

MEGAWHOP cloning: a method of creating random mutagenesis ... - PubMed

WebTherefore, we designed our mutants library construction method based on error-prone PCR and MEGWHOP ( megaprimer PCR of whole plasmid) cloning [1]. This method has high efficiency and high positive fate compared with traditional methods. Please click here to view our detailed protocol. WebThe present method, which we designated MEGAWHOP (megaprimer PCR of whole plasmid), is thus ideal for creating random mutagenesis megalibraries. Publication types …

Megawhop pcr

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Web3 mei 2024 · We established a PobR mutant library with a capacity of 550,000 mutants using error-prone PCR and Megawhop PCR. Through our screening, two mutants were … Web1 dec. 2002 · The PCR product used as megaprimer was purified and cloned into the pET-30a (+) vector by the megaprimer PCR method of whole plasmid (MEGAWHOP) as …

WebA modified polymerase chain reaction (PCR) was developed to introduce random point mutations into cloned genes and was used to mutagenize the gene that encodes the … Web3 okt. 2024 · Make sure that the template of MEGAWHOP PCR is fresh to improve the construction efficiency. Error-p rone PCR 1 Add the following reagent to a PCR tu be (50 μ l) (Random Muta genesis Kit by ...

Web17 sep. 2024 · The resultant libraries were virtually free from species with no inserts or multiple inserts. The present method, which we designated MEGAWHOP (megaprimer … Web17 feb. 2015 · • MEGAWHOP PCR/ QuickChange PCR • DNA Sequencing • Research analysis and reports • Present research data • SDS-PAGE/ Western Blots • HEK293 cell culture • Co-immunoprecipitation

WebThe double-stranded product A-M is purified and used as a primer (hence the name “megaprimer” ref. 1) in the second PCR (PCR-2) together with the other flanking primer …

WebThis megaprimer is purified and then used as primer with the template plasmid in a second PCR whole plasmid (Megawhop) reaction. This PCR product is cleaned up and … toddlers trampoline argosWebCreating random mutagenesis libraries by megaprimer PCR of whole plasmid (MEGAWHOP) Methods Mol Biol. 2003;231:23-8. doi: 10.1385/1-59259-395-X:23. … pentre berw angleseyWeb1 jan. 2010 · This technique known as Megaprimer PCR of whole plasmid (MegaWHOP) (Fig. 3) allows the ligation-free cloning of mutated genes into plasmids and their … pentre bach wrexhamWebMegawhop technique. 1. The template plasmid with the gene coding for C. violaceum ω TA with F88A/A231F mutations was first denatured in the PCR program by increasing the … toddlers \\u0026 tiaras tv showWeb31 dec. 2011 · The MEGAWHOP PCR was achieved by using PfuS DNA polymerase (2.5 U), PfuS DNA polymerase buffer (1X), plasmid DNA template (50 ng), dNTP mix (0.2 … toddlers\\u0027 burton 2l one pieceWebThe monoamine oxidase mutant A-1 (F210V/L213C) from Aspergillus niger showed some catalytic activity on mexiletine. To futher improve its activity, the mutant was subjected to directed evolution with MegaWHOP PCR (Megaprimer PCR of Whole Plasmid) and selection employing a high-throughput agar plate- … pentrebychan crematorium addressWeb8 okt. 2014 · The PCR product was used as a megaprimer to perform MEGAWHOP PCR (Miyazaki 2011) using pET-pel R227W plasmid as template. Following the MEGAWHOP PCR, the PCR products were digested with DpnI and transformed into E. coli. All clones from the libraries were subjected to downstream high-throughput screening. Site-directed … pentrebach wales