Touchdown pcr steps8/28/2023 ![]() ![]() Overlap extension PCR: an efficient method for transgene construction. A rapid and convenient variant of fusion-PCR to construct chimeric flaviviruses. Biotechniques, 1990, 8(4):404-407Ĭharlier N, Molenkamp R, Leyssen P, et al. The “megaprimer” method of site-directed mutagenesis. A rapid method for the construction of synthetic genes using the polymerase chain reaction. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Precise gene disruption in Saccharomyces cerevisiae by double fusion polymerase chain reaction. Chinese Journal of Biochemistry and Molecular Biol, 2012, 28(4): 375-379.Īmberg D C, Botstein D, Beasley E M. ![]() Quadruple DNA Fragments Fusion using Touchdown-Overlap Extension PCR. Supported by Science and Technology Development Program of Henan Province (No.082102150048)ĬHAI Ran,SUN Qiang,QIU Li-You. Gene fusion overlap extension PCR touchdown PCR homologous recombination The results suggested that TD-OE PCR could be an effective method to prepare long multiple fragment fusions for the research in somatic cell knockout/in or partial fungal genome synthetic applications. A 98.5% consensus between the fusion product and the four separate DNA fragments was detected. The desired fusion product of 7.05 kb was obtained through the overlap PCR with one round, and then cloned into a T-vector and sequenced. In step II, the annealing temperature were decreased by 0.5 ℃from 60 ℃ to a touchdown at 56 ℃ with the supply of LA DNA polymerase. In step I, the annealing temperature were decreased by 0.5 ℃from 61.5 ℃ to a touchdown at 57.5 ℃ starting from the second cycle. In an attempt to construct a homologous recombination fragment to replace the glucan synthase promoter in Pleurotus ostreatus, four fragments of 1 015, 2 822, 2 206 and 1 008 bp in length were used for the fragment fusion. Abstract Here we introduced a genes fusion procedure using touchdown PCR (TD PCR) in the step I and II of classic overlap extension PCR (OE PCR) to reduce the inappropriate annealing and the extra 3′-end adenine which added by Tag DNA polymerase. ![]()
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