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
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