Final concentration of reactions consisted of 0.6 [mu]M forward and reverse primer, 0.4 [mu]M BSA, 2-20 ng DNA, 12.5 [mu]L 2xEconoTaq (Lucigen
Corporation, Middleton,WI, USA), and 7.5 [mu]L PCR grade water.
Subsequently, a genotype PCR assay for the MGL2-09 sample was performed by using 9 additional thermostable DNA polymerases either with 3'-5' exonuclease activity ("proofreading") or without 3'-5' exonuclease activity ("nonproofreading"), including KAPA2G Robust HotStart (KAPA Biosystems), Klentaq1 (DNA Polymerase Technology, St Louis, Missouri), AmpliTaq Gold (Thermo Fisher Scientific), Platinum Taq (Thermo Fisher Scientific), FailSafe PCR Enzyme Blend (Lucigen
Corp, Middleton, Wisconsin), TaKaRa LA Taq (Takara Bio USA, Mountain View, California), Platinum Taq High Fidelity (Thermo Fisher Scientific), Q5 High-Fidelity (New England Biolabs, Ipswich, Massachusetts), and Deep Vent DNA Polymerase (New England Biolabs).
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All PCR reactions were performed with EconoTaq DNA Polymerase (Lucigen
, Middleton, WI) according to the manufacturer's instructions, with an annealing temperature of 56 [degrees]C.
Eg AgB8/1 protein was expressed in [p.sup.Rham] vector in frame with solubility enhancing sumofusion protein (Expresso[TM] Rhamnose cloning and expression kit; Lucigen
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To amplify the DNA a single-step 30-cycle PCR using EconoTaq PLUS 2x Master Mix (Lucigen
, Meddleton, WI) were used under the following conditions: 94[degrees]C for 2 minutes, followed by 30 cycles of 95[degrees]C for 120 seconds; 42[degrees]C for 30 seconds and 72[degrees]C for 4 minutes, after which a final elongation step at 72[degrees] C for 120 minutes was performed.
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EconoTaq DNA polymerase was purchased from Lucigen
First strand cDNA was synthesized by reverse transcriptase on 2 pg of total RNA using MULV reverse transcriptase enzyme (Lucigen
, USA) with an oligo-dT primer.
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Amplifications were conducted in 50 [micro]L final reaction volumes, each containing standard PCR Buffer with 1.5 mM Mg[Cl.sub.2] (Innis & Gelfand 1990), 2 [micro]L DNA template, 50 ng of each primer, 125 [micro]M of each dNTP and 1 U EconoTaq DNA polymerase (Lucigen
Corp., Middleton, WI).