The spike-in control, including 3 distinct lambda DNA amplification products (~180 bp; one without modification and the other two with 5mC and 5hmC modifications), was prepared as follow47: lambda DNA was PCR amplified by Taq DNA Polymerase (NEB) and purified by AMPure XP beads (Beckman...
To simultaneously improve product yield and amplification fidelity,Tba5 plus DNA polymerase mixtures were constituted with various amounts ofTba5 DNA polymerase mixed withTaqDNA polymerase. TheTba5 plus DNA polymerase mixtures robustly amplified up to 25-kb λ DNA fragments. In addition, the PCR ...
Total RNA isolation and complementary DNA synthesis Total RNA was extracted using the Nucleospin® RNA Plant kit (Macherey–Nagel, Düren, Germany), starting from 100 mg of finely grinded leaf samples, according to the manufacturer’s instructions. RNA concentration was estimated by reading spectrop...
PCR was carried out in 25 µl triplicate reactions using 2 µl genomic DNA (100× dilution of original extract), 12.5 µl of GoTaq Green Master Mix (Promega), 2 µl of 10 µM 13-bp tagged forward and reverse primers, 1 µl of bovine serum albumin (1 mg/ml; New Englan...
FastTaq Premix(with dye) 10 *1 mL 50 mL 1,000 mL FastPfu DNA polymerase 100 U 500 U 1,000 U FastPfu Premix 1 mL 10*1 mL 50 mL 1,000 mL FastPfu Premix (with dye) 1 mL 10*1 mL 50 mL 1,000 mL P6 High-Fidelity polymerase 100 U 500 U 1,000 U P6 High-Fidelity Premix 1...
(NH4)2SO4, 3 mmol l − 1 MgCl2, 50 μmol l − 1 each dNTP, 480 nmol l − 1 of each specific primer for SEA deletion, 900 nmol l − 1 of each primer for SNP (rs3760053), 1 μmol l − 1 SYBR green and 0.02 units Taq DNA polymerase (Biolabs Co., Ltd., Ipswich,...
For the pre- and post-capture PCR reaction, we tested the performance of Tks Gflex™ DNA Polymerase (Takara Bio Inc., Japan) and LongAmp™ Taq 2× Master Mix (New England Biolabs, Ipswich, MA, USA) for 1000 bp fragment amplification. The use of LongAmp™ Taq with PRM primers incl...
DNA analysis for deletional α-thalassaemia The five common deletions causing α-thalassaemia in Southeast Asia: -α3.7, -α4.2, –SEA, –FIL, –THAIwere characterised using an adapted multiplex-PCR21(Supplementary Table 1). Hot StartTaqDNA polymerase (Qiagen HotStarTaqPlusDNA Polymerase, Hilden,...
For each sample, two duplex PCRs were carried out using 5 ng genomic DNA and 0.5 U Taq DNA polymerase (NEB) in a buffer with final concentrations of 50 mM Tris–HCl pH8.8, 12.5 mM ammonium sulphate, 1.4 mM magnesium chloride, 7.5 mM 2-mercaptoethanol, 125 μg/ml BSA and 200 μM ...
PCR-based validation Presence or absence of inserts was checked by polymerase chain reaction (PCR) amplifications using the 2X Power Taq PCR mix (Bioteke Corp.) according to manufacturer's instructions and using primers designed within the Tnt1 sequence (5′ and 3′ sides of the Tnt1 ...