Reactions (50 μl) containing 100 ng of Substrate A and 100 ng Substrate B, either unmodified or fully 5hmC-modified, 15.4 μg LiNE, 20 mM HEPES (pH 7.9), 1 mM EDTA pH 8.0, 3 mM MgCl2, 10 mM KCl, 10 mM 2-mercaptoethanol, 4% (v/v) glycerol, were incubated for 15 min at 37°C. Reactions were terminated by the addition of 10 μl stop solution (50 mM Tris (pH 8.0), 2% (w/v) SDS, 100 mM EDTA (pH 8.0) and 1 μg Proteinase K) and incubated at 42°C for at least 30 min. DNA was purified using a Qiagen PCR clean kit. Two qPCR reactions were run on each reaction: control region and recombined region (primers in Supplementary Table S2) were completed as previously described (40 ). The percent recombinant was computed using the formula: ((recombined region quantity)/(control region recovered)) × 100%. The value recorded for the reaction incubated in the absence of LiNE was subtracted from the result of this calculation.
Pcr clean kit
Manufactured by Qiagen
The PCR clean kit is a laboratory equipment product designed to purify and clean PCR (Polymerase Chain Reaction) samples. It facilitates the removal of unwanted components from PCR reactions, such as primers, nucleotides, and enzymes, to prepare the samples for further analysis or downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Sc 81405, by Santa Cruz Biotechnology (1 mentions)
Sc 81406, by Santa Cruz Biotechnology (1 mentions)
Sc 292119, by Santa Cruz Biotechnology (1 mentions)
Abi 3730xl, by Thermo Fisher Scientific (1 mentions)
Hifi hotstart readymix, by Roche (1 mentions)
Iplex gold assay, by Labcorp (1 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Massarray analyzer, by Labcorp (1 mentions)
Megashortscript t7 kit, by Thermo Fisher Scientific (1 mentions)
Gtf3c2, by Santa Cruz Biotechnology (1 mentions)
5 protocols using pcr clean kit
1
Enzymatic DNA Recombination Assay
2
ChIP-qPCR Assay for Transcription Factors
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HepG2 cells with STAT3 or TP73 silencing and their control cells were cultured in 10 cm dishes. At 85% confluence, cells were fixed for 10 min using 10 mL of 1% formaldehyde solution freshly prepared with 1×PBS and subsequently quenched with 1 mL of 2.5 M glycine solution. After washing three times with 1×PBS solution, cell samples were harvested with cell scrappers and were sheared with an ultrasonication machine (Scientz 98-III,Ningbo, China). After sonicating for 20 min (pulse 5 s and interval 5 s) with 25% power, cell lysate was centrifuged for 10 min at 12,000 rpm, and the resulting supernatant was retained for ChIP assays as described previously (Zhang et al., 2022 (link)), where antibodies against TBP (SC-204; Santa Cruz Biotech.), BRF1 (SC-81405; Santa Cruz Biotech.), GTF3C2 (SC-81406; Santa Cruz Biotech.), and POL3RA (SC-292119, Santa Cruz Biotech.) were used in ChIP. The DNA from ChIP assays was purified with a QIAGEN PCR clean kit and eluted with 40 μL ddH2O. 1 μL of ChIP DNA sample (1/40) was used for a qPCR reaction, while 0.02% input DNA (0.1 ng genomic DNA) was used in a qPCR reaction for positive control. Relative enrichment was obtained by comparing the quantity of target DNA in 1 μL of ChIP DNA sample to that in 0.02% input DNA.
3
Genotyping FcγR Polymorphisms in Peripheral Blood
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Blood samples were collected before treatment. Archived peripheral blood mononuclear cells were used for DNA extraction with the QIAamp DNA blood mini kit (Qiagen). Nested polymerase chain reaction (PCR) was conducted to detect single-nucleotide polymorphisms in FcγRIIA and/or IIIA using the same primers as in a previous study.21 (link) PCR was carried out using the HiFi HotStart ReadyMix (KAPA Biosystems) and optimized protocols. The PCR products were purified using the PCR clean kit (Qiagen) and then sequenced on an ABI3730XL (Applied Biosystems) with the BigDye Terminator v3.1 Cycle Sequencing Kit. The PCR products of 110 samples were also analyzed on MassARRAY analyzer (Sequenom) with the iPLEX Gold assay (Sequenom) using primers, as in a previous study,21 (link) to identify the A559C polymorphism in FcγRIIIA and the A519G polymorphism in FcγRIIA.
4
Generating PCR Primers and sgRNA
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The PCR primers are listed in Supplementary Table S1 . The PCR primers were annealed, extended by 3 cycles of PCR reactions (95°C 2 min, 50°C 2 min, and 72°C 2 min) and stored at 10°C. The sgRNA primers were designed to contain a T7 promoter and overlap with Cas9 guide-constant sequence (Talbot and Amacher 2014 (link)). After the PCR fragment was purified by a QIAGEN PCR clean kit, the sgRNA was generated by in vitro transcription at 37°C for 2 h using a MEGAshortscript T7 Kit (AM1354, Thermo Scientific). After digestion of dsDNA template by 1 μl Turbo DNase 37°C at 15 min, sgRNA was precipitated and purified.
5
ChIP-qPCR Analysis of Transcription Factor Binding
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The 293T cell line stably expressing GATA4 shRNA and its control cell line were cultured in dishes of 10 cm. At 90% confluence, cells were fixed for 10 min using 10 ml 1% formaldehyde freshly prepared with 1 × PBS solution, followed by quenching with 1 ml of 2.5 M glycine solution. After washing twice with 1 × PBS solution, the cells were harvested and disrupted with an ultrasonicator. The samples disrupted were subject to centrifuging for 10 min at 12,000 rpm. The supernatant was retained and used for ChIP assays as described previously (48 (link)), where TBP (catalog no.: SC-204; Santa Cruz Biotechnology, Inc), BRF1 (catalog no.: SC-81405; Santa Cruz Biotechnology, Inc), GTF3C2 (catalog no.: SC-81406; Santa Cruz Biotechnology, Inc), and POL3RK (catalog no.: Ab57214; Abcam) antibodies were used for the assays. After chromatin decrosslinking, DNA was purified from the ChIP samples using a Qiagen PCR Clean kit and eluted with 40 μl double-distilled water. One microliter of the DNA sample obtained from the ChIP assay for individual factors was used for a qPCR, where 0.1 ng genomic DNA (equal to 0.01% input) was used as a positive control. Relative occupancy was calculated with the quantity of promoter DNA in 1 μl of ChIP sample divided by that in 0.1 ng genomic DNA.
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