Genomic DNA was extracted from DF-1 cells after puromycin selection. Genomic regions encompassing the CRISPR/Cas9 target sites were amplified using specific primer sets. PCR analysis of the targeted loci was examined in a total volume of 20 μL containing 100 ng genomic DNA, 10× PCR buffer (BioFACT, Daejeon, Korea), 0.4 μL dNTPs (10 mmol/L each), 10 pmol of each primer, and 0.5 U Taq polymerase (BioFACT) under the following thermocycling conditions: 2 min at 95 °C, followed by 35 cycles of 20 s at 95 °C, 40 s at 65 °C, and 30 s at 72 °C, and a final 5 min at 72 °C. Primers are listed in Table 1 . The PCR amplicons were re-annealed to form a heteroduplex DNA structure after denaturation. Subsequently, the heteroduplex amplicons were treated with 5 units T7E1 endonuclease (New England Biolabs) for 20 min at 37 °C and then analyzed by 1% agarose gel electrophoresis.
Taq polymerase
Manufactured by Biofact
Taq polymerase is a thermostable DNA polymerase enzyme derived from the bacterium Thermus aquaticus. It is a widely used enzyme in molecular biology laboratories for various DNA amplification techniques, such as polymerase chain reaction (PCR).
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Lab products found in correlation
2 protocols using taq polymerase
1
Targeted CRISPR/Cas9 Genome Editing Analysis
2
Surface Functionalization of Polymers
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(3-Aminopropyl)triethoxysilane (APTES; 99%) and [2-(3,4-epoxycyclohexyl)ethyl]trimethoxysilane (ECTMS; 98%) were purchased from Sigma-Aldrich. The PDMS prepolymer (Sylgard 184) and curing agent were obtained from Dow Corning. Poly(methyl methacrylate) (PMMA), polycarbonate (PC), polystyrene (PS), and polyethylene terephthalate (PET) were purchased from Goodfellow. Fluospheres amine (0.2 μm, red) and TE buffer (10 nM Tris–HCl, 0.1 nM EDTA, pH 8.0) were obtained from Thermo Fisher Scientific. Taq polymerase, PCR buffer solutions, and dNTPs were obtained from BioFact (Daejeon, Korea). Agarose powder was purchased from BioShop (Ontario, Canada). A 100 bp DNA size marker was purchased from Takara (Shiga, Japan). Ethidium bromide (EtBr)-based dye (Loading STAR) was purchased from Dynebio (Seongnam, Korea).
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