Genomic DNA was isolated from pcDNA3.1/myc-His(B)-HBx or empty vector transfected Huh7 cells using DNA extraction kit (FAVORGEN Biotech Corp., Taiwan). 2ug of isolated genomic DNA was bisulfite treated and purified using EZ DNA methylation kit (Zymo Research, CA, USA). The bisulfite-modified genomic DNA was amplified with bisulfite specific primers of USF-1 promoter (Additional file 1 : Table S2) for 35 cycles of 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 20 s and the amplified products were cloned into pJET1.2 blunt vector. Six different clones from each group pcDNA3.1/myc-His(B)-HBx /empty vector transfected cells were screened by sequencing and DNA methylation status was analyzed.
Dna extraction kit
Manufactured by Favorgen Biotech
Sourced in Austria
The DNA extraction kit is a laboratory tool designed to isolate and purify DNA from various biological samples. It consists of a set of reagents and protocols that enable the efficient extraction and recovery of DNA for further analysis and experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Ez dna methylation kit, by Zymo Research (1 mentions)
Chromatin immunoprecipitation chip assay kit, by Merck Group (1 mentions)
Fermentas pcr kit, by Thermo Fisher Scientific (1 mentions)
Applied biosystems 3130xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Dynazyme ext dna polymerase, by Thermo Fisher Scientific (1 mentions)
Abi veriti, by Thermo Fisher Scientific (1 mentions)
Nanophotometer pearl, by Implen (1 mentions)
Pcr clean up gel extraction nucleospin gel, by Macherey-Nagel (1 mentions)
Pcr clean up kit, by Macherey-Nagel (1 mentions)
12 protocols using dna extraction kit
1
Bisulfite Sequencing of USF-1 Promoter
2
FOXO1 ChIP-PCR Profiling
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Cells transfected with empty vector or vectors encoding FOXO1-Flag or FOXO1-Delta DB were subjected to chromatin immunoprecipitation experiments performed with the Chromatin Immunoprecipitation (ChIP) Assay Kit (Millipore), following the manufacturer's instructions. Anti-Flag and control anti-GAPDH antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and DNA was extracted using DNA extraction kit (Favorgen biotech corp., Taiwan). PCR reactions were conducted using the Fermentas PCR kit (Thermo scientific, PA, USA), following the cycling conditions: 1 cycle (95°C 2 min), 30 cycles (95°C 30 s, 58°C 30 s, 72°C 1 min), and 1 cycle (72°C 5 min) with the indicated primers. Migration of PCR products was performed in 2% agarose gels.
3
UCP2 -866G/A Genotyping by PCR-RFLP
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The DNA sample was isolated from buffy coat using a commercial DNA extraction kit (Favorgen, Pingtung City, Taiwan). UCP2 −866G/A genotyping was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with forward primer: 5′-CACGCTGCTTCTGCCAGGAC-3′ and reverse primer: 5′-AGGCTCAGGAGATGGACCG-3′. PCR conditions are: 8 min of denaturation in 95 °C followed by 35 cycles of 95 °C for 1 min (denaturation), 55 °C for 1 min (annealing), 68 °C for 1 min (extension) and 72 °C for 7 min (final extension). The PCR product then digested using BST UI enzyme digestion. Restriction fragments were resolved on a 3% agarose gel.
4
Saliva DNA Extraction and DGGE Analysis
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One ml of saliva was dissolved in 4ml PBS and DNA was extracted based on instruction of DNA extraction kit (FavorGen) and was stored at -20°C. Since the PCR product were subsequently applied for DGGE, at the start of forward primer, a GC clamp with 40 bases was added. Primers sequences for 16S rDNA are shown in Table 1 (Table 1 ). The thermal cycling was as follow: a first denaturation step: 94°C in 1 min followed by 30 cycles of denaturation at 94°C for 45 seconds, annealing at 53°C in 30sec, extension at 72°C for 35 seconds and a round of 5 min at 72°C as the final extension process. The PCR mixture contained 2X master mix (YektaTajhizAzma), 10pmol/ μl of each primer and 2 μl (50ng) DNA template. Accuracy of PCR reaction was characterized by agarose gel electrophoresis. At the next step, PCR products were separated by DGGE Electrophoresis.
5
Genetic Polymorphism Analysis in Blood
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Peripheral blood sample was drawn to examine gene polymorphisms. All the patients’ genomic DNA was extracted from 200 µl of blood sample in EDTA by using a DNA extraction kit (FAVORGEN, Ping-Tung, Taiwan). Polymerase chain reactions (PCRs), PCR-restriction fragment length polymorphisms (RFLPs), or direct sequencing were performed to identify polymorphisms of TPH1 rs211105 (11:g.18033757T>G) located within intron 3, rs4537731 (11:g.18047335A>G) located within the promoter region at –1066, insertion/deletion polymorphisms in 5-HTTLPR, and VNTR counts in 5-HTT, as previously reported.(24 (link)) The specimens for direct sequencing were run on an Applied Biosystems 3130xl Genetic Analyzer (Applied Biosystems, Invitrogen Life Technologies, Carlsbad, CA), conforming with the manufacturer’s recommendations.
6
DNA Extraction and Bisulfite Conversion
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DNA was extracted using DNA extraction kit (Favorgen
Biotech, Austria) as manufaturer’s protocol. Yield and
purity of DNA were determined using a NanoDrop
spectrophotometer at 260/280 nm (NanoDrop ND-2000C
Spectrophotometer, Thermo Fisher Scientific, USA).
Then, DNA was treated with sodium bisulfite using
Fast EpiTect Kit (Qiagen, USA). The treated DNA was
resuspended in water and stored at -80˚C until the next
steps.
Biotech, Austria) as manufaturer’s protocol. Yield and
purity of DNA were determined using a NanoDrop
spectrophotometer at 260/280 nm (NanoDrop ND-2000C
Spectrophotometer, Thermo Fisher Scientific, USA).
Then, DNA was treated with sodium bisulfite using
Fast EpiTect Kit (Qiagen, USA). The treated DNA was
resuspended in water and stored at -80˚C until the next
steps.
7
UV-Induced DNA Fragmentation in RA-FLSs
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RA-FLSs were cultured in 6 cm dishes to a confluence of 60–80%. The cells were then exposed to UV light for 5, 10, and 15 min. The medium was removed and washed twice with PBS for the following experiment. RA-FLSs were harvested for DNA extraction using a DNA extraction kit (Favorgen). DNA fragmentation was performed by ultrasonication for 30 min to shear the DNA into fragments of 500 bp as determined by DNA gel electrophoresis. RA-FLSs cultured in 6-well plates were transfected with 5 μg of DNA fragments for 24–72 h in the absence or presence of Lipofectamine 3000. Furthermore, another group of RA-FLSs was also transfected with different concentrations of DNA fragments (1, 5, and 10 μg) with Lipofectamine 3000 for 24 h. All the medium was removed, and the cells were washed once with PBS before the next step of analysis.
8
Integration Junction PCR for Transgene Detection
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Integration junction PCR was performed on 200 ng of genomic DNA (DNA extraction kit; Favorgen Biotech Corp., Ping-Tung, Taiwan) using DyNAzyme EXT DNA polymerase (Thermo Scientific, Rockford, IL, USA). Left integration junction PCR (454-bp amplicon) was performed using primer pairs specific to a locus on chromosome 8p22 (forward: 5′-GGGCTCTGGAGTAAAGGTGAAA-3′) and donor vector (reverse: 5′-GTTCGCCGGGATCAACTACC-3′). Right integration junction PCR (333-bp amplicon) was performed using primer pairs specific to the vector sequence (forward: 5′-TCGACGATGTAGGTCACGG-3′) and chromosome 8p22 genomic DNA (reverse: 5′-GCATGGCCTCATTTCCGTCT-3′). Transgene-specific PCR was performed with intron-spanning primers specific to the C1-domain of the integrated hybrid FVIII cDNA (forward: 5′-AGCTTTCAGAAGAGAACCCGACAC-3′ reverse: 5′-TCCCAGGGGAGTCTGACACTTCTTGCTGTACACCAGGAAAGT3′). Control genomic PCR (900-bp amplicon) was performed with genomic primers (forward: 5′-AAGAAGCGCACCACCTCCAGGTTCT-3′ reverse: 5′-ATGACCTCATGCTCTTGGCCCTCGTA-3′). Amplified products were electrophoresed on 1% agarose gels and imaged using BioRad Gel Doc 2000 transilluminator (Bio-Rad Laboratories, Hercules, CA, USA).
9
DNA Extraction from Whole Blood
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Fresh blood (3 mL) was drawn from all the individuals in Ethylenediamine tetraacetic acid tubes. The blood was stored at 4 °C until the extraction of genome. DNA was extracted from 200 μL of whole blood by Favorgen DNA extraction kit, according to the manufacturer's instructions. Isolated DNA samples were quantified by UV spectrophotometry at 260 nm absorbance and diluted with distilled water to the concentration required for polymerase chain reaction (PCR) (100 ng/μL).
10
Meat DNA Extraction and Electrochemical Detection
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Total DNA from meats was isolated using a DNA extraction kit (Favorgen Biotech Corp, Taiwan) following manufacturer instructions. A UV–vis spectrophotometer (NanoPhotometer Pearl, Implen GmbH, Germany) was used to assess the extracted DNA content and purity at an absorbance of 260 nm and at an absorbance ratio of A260/A280 [19 (link)]. The extracted DNA was heated at 95 °C for 8 min in the PCR machine (ABI Veriti, Applied Biosystems) to denature the dsDNA into ssDNA. It was then immediately dropped onto an SPCE-modified probe and detected by using DPV as stated in previous sections.
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