Thermo Fisher
Phusion High-Fidelity DNA Polymerase
F530L
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This polymerase has such a low error rate it is unquantifiable. Used for annealing reactions between 67-72 C, and extension at 72 C.
PCR for cloning
cDNA generated from Superscript IV kit
dH2O 37 ul5x Phusion HF buffer (use GC buffer if >75%) 10 uldNTP (10mM each dNTP, final 200uM) *must be PCR grade 1 ulPrimer #1 (100uM, final 0.5uM) 0.25 ulPrimer #2 (100uM, final 0.5uM) 0.25 ulTemplate (1pg-10ng plasmid, 50-250ng gDNA) 1ul(DMSO, optional for GC-rich amplicons) (1.5ul)Phusion DNA polymerase 0.5ul (1U)Total 50 ulRun a standard PCR cycler to 37 cycles.
Run firsts ample on a gradient to ascertain highest Tm that works
This enzyme has been used to clone 86 genes and has resulted in zero errors when followed by conventional restriction cloning.
N/A
Amazing product, works every time with good primers
Cost? Not really a drawback.
Probably the best commercial product for cloning.