Plasmid pUC19 DNA linearized with BamHI (TaKaRa) and purified with a QIAquick PCR kit (Qiagen) was stored at −20°C. Calf thymus DNA (Sigma) solubilized in 10 mM Tris–1 mM EDTA (pH 8) was sonicated to reduce the average DNA fragment size to ~1,000 bp and was stored at +4°C. Plasmid or thymus DNA was exposed to bacteria as follows: in a 96-well plate or in 1.5-ml Eppendorf microtubes, 400 ng (or the indicated amount) of DNA was added to 100 µl DMEM–25 mM HEPES (Invitrogen) inoculated with 3 × 106 bacteria pregrown to the exponential phase or in the population numbers given in the text. As controls, DNA was treated with 5 mM MMS (Sigma), 80 µM cisplatin (Sigma), or 150 µM MMC (Sigma) activated with 5 mM dithiothreitol (DTT). Following 4 h at 37°C, bacteria were pelleted and the DNA was purified from the supernatant using a Qiagen QIAquick PCR kit. For wild-type enterobacterial strains that release nucleases, the bacteria were grown in DMEM–25 mM HEPES without the target DNA for 3.5 h, and then EDTA (1 mM) and target DNA were added and the mixture was incubated for 40 min at 37°C.
Qiaquick pcr kit
Manufactured by Qiagen
Sourced in Germany, United States
The QIAquick PCR kit is a laboratory tool designed for the purification of PCR (Polymerase Chain Reaction) amplicons. It utilizes a silica-membrane based system to efficiently capture and purify DNA fragments from PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 4000, by Illumina (4 mentions)
Hiseq 2500 platform, by Illumina (4 mentions)
Proteinase k, by Thermo Fisher Scientific (3 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (3 mentions)
Api 50 chl kit, by bioMérieux (3 mentions)
Genomic dna preparation kit, by Promega (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Hiseq 2000, by Illumina (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Bioruptor pico, by Diagenode (2 mentions)
Microplex library preparation kit, by Diagenode (2 mentions)
Ribo zero magnetic kit, by Illumina (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Smad2, by Abcam (2 mentions)
Protein g magnetic dynabeads, by Thermo Fisher Scientific (2 mentions)
Calf thymus dna, by Merck Group (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Bamhi, by Takara Bio (1 mentions)
Faststart universal sybr green, by Roche (1 mentions)
38 protocols using qiaquick pcr kit
1
Bacterial DNA-Binding Assay Protocol
2
ChIP-qPCR Analysis of Transcription Factors
3
ChIP Assay with Protein G Beads
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To perform ChIP assays, 1% formaldehyde for 10 min was used to cross-link the proteins to the DNA, then the reaction was quenched with 0.125 M glycine. After nuclei lysis, the lysates were sonicated and the immunoprecipitation was carried out using Dynabeads Protein G (Invitrogen, cat. 10004D). 0,1 mg/mL RNase A (Thermo Scientific) and Proteinase K (20 mg/mL, Thermo Scientific) were used to reverse the cross-links. The DNA was purified by QIAquick PCR kit (QIAGEN). The DNA levels were measured by real-time quantitative PCR. ChIP was performed with the following antibodies: anti-p53 (Leica, cat. P53-CM5P-L), and mouse IgG Isotype control (Invitrogen, cat. 10500C).
4
RNA Extraction and Sequencing Pipeline
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Total RNA was extracted from the experimental samples using the TRIzol (Invitrogen, Carlsbad, CA, USA) kit according to the instructions; RNA purity and concentration were preliminarily detected using a NanoDrop2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA), and RNA integrity was accurately quantified using an Agilent 2100 (Agilent Technologies, Santa Clara, CA, USA) bioanalyzer.
Then, we removed the rRNAs from the total RNA of the sample, retained the mRNAs and ncRNAs, reverse transcribed the obtained RNA, purified the cDNA fragment using the QiaQuick PCR kit (Qiagen, Venlo, Holland), repaired the end, added PolyA, added sequencing linker, degraded the product by UNG (Uracil-N-Glycosylase) enzyme and amplified the product by PCR, and sequenced the library by Illumina HiSeqTM4000. The raw data on the machine were subjected to quality control and data filtering, and the processed reads were aligned to the pig reference genome (release Sscrofa11.1) using HISAT2 [21 (link)]. Differentially expressed genes were identified by DESeq2 [22 (link)], while differentially expressed genes were validated by qPCR (Table S2 ).
Then, we removed the rRNAs from the total RNA of the sample, retained the mRNAs and ncRNAs, reverse transcribed the obtained RNA, purified the cDNA fragment using the QiaQuick PCR kit (Qiagen, Venlo, Holland), repaired the end, added PolyA, added sequencing linker, degraded the product by UNG (Uracil-N-Glycosylase) enzyme and amplified the product by PCR, and sequenced the library by Illumina HiSeqTM4000. The raw data on the machine were subjected to quality control and data filtering, and the processed reads were aligned to the pig reference genome (release Sscrofa11.1) using HISAT2 [21 (link)]. Differentially expressed genes were identified by DESeq2 [22 (link)], while differentially expressed genes were validated by qPCR (
5
Genotyping-by-Sequencing for SNP Discovery
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To obtain SNPs, genomic libraries were developed using the genotyping-by-sequencing technique (GBS) with two restriction enzymes, according to the protocol described by Poland et al. [32 (link)] with modifications. First, 7 μl of genomic DNA from each sample was digested at 37°C for 12 h using the restriction enzymes NsiI and MspI. Subsequently, 0.02 μM barcode-specific adapters for Illumina technology were ligated to the ends of the digested fragments. Binding reaction was performed at 22°C for 2 h, 65°C for 20 min, 10°C indefinitely. After the adapters were ligated, the samples were purified using a QIAquick PCR Kit (Qiagen). The library was enriched by PCR (Polymerase Chain Reaction) using the following amplification program: 95°C for 30 s, followed by 16 cycles of 95°C for 10 s, 62°C for 20 s, and 72°C for 30 s, and ending at 72°C for 5 min. Finally, the library was purified using a QIAgen® QIAquick PCR Purification Kit. The Agilent DNA 12000 kit and Agilent® 2100 Bioanalyzer System were used to verify the average size of the DNA fragments. Sequencing was performed using the Illumina® HiSeq 2500 Mid Output Kit v4 (50 cycles) (Illumina Inc., San Diego, CA, USA) in a single-end configuration.
6
Differential Expression Analysis of CircRNAs
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To identify differentially expressed circRNAs, the left lungs of sham (n = 3) and CLP (n = 3) mice were used for high-throughput sequencing (Nanjing Decode Genomics Center, Nanjing, China) (Figure 1 ). Total RNA was extracted and ribosomal RNA was removed. A strand-specific RNA library was constructed. RNAs were digested into 200–500 bp fragments and then used as templates to synthesize cDNAs in the presence of a six-base random primer, dNTPs, RNase H (Thermo Scientific, USA), DNA polymerase I (New England Biolabs Inc., USA), and buffer (Thermo Scientific, USA). The PCR products were purified using the QiaQuick PCR kit (QIAGEN, GER) and end-repaired, followed by A-tail addition and sequencing-linker ligation. Fragment sizes were determined by agarose gel electrophoresis and then PCR amplification was performed. The sequenced library was used for Sanger sequencing by adopting a double-ended sequencing strategy.Figure 1 ![]()
http://www.bioconductor.org/packages/release/bioc/html/edgeR.html ).
Flow chart.
7
Bacterial Identification Using API 50 CHL and 16S rDNA
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The API 50 CHL kit (Biomerieux, Rue des Aqueducs, France) was used as a simple
way of identifying the isolated bacterial strains by measuring sugar
utilization. Colonies cultured in MRS broth were inoculated into the API 50 CHL
kit and incubated at 37°C. After 24 h and 48 h, the change in color
(yellow) was measured according to the type of sugar. The measurements were used
for simple identification of the bacteria via the Biomerieux DB (https://apiweb.biomerieux.com ).
In addition, 16S rDNA was analyzed for genetic identification. Specifically,
after extracting genomic DNA using a genomic DNA preparation kit (Promega,
Madison, WI, USA), a polymerase chain reaction (PCR) reaction was run using the
universal primer pair 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and
1492R (5′-TAC GGY TAC CTT GTT ACG ACT T-3′) to amplify the 16S
rDNA (Buck and Gilliland, 1994 (link)). The PCR
products were purified using a QIA quick PCR kit (QIAGEN, Hilden, Germany) and
sequencing was outsourced to Macrogen (Seoul, Korea). The base sequences were
then compared with the NCBI GenBank DB using BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi ).
way of identifying the isolated bacterial strains by measuring sugar
utilization. Colonies cultured in MRS broth were inoculated into the API 50 CHL
kit and incubated at 37°C. After 24 h and 48 h, the change in color
(yellow) was measured according to the type of sugar. The measurements were used
for simple identification of the bacteria via the Biomerieux DB (
In addition, 16S rDNA was analyzed for genetic identification. Specifically,
after extracting genomic DNA using a genomic DNA preparation kit (Promega,
Madison, WI, USA), a polymerase chain reaction (PCR) reaction was run using the
universal primer pair 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and
1492R (5′-TAC GGY TAC CTT GTT ACG ACT T-3′) to amplify the 16S
rDNA (Buck and Gilliland, 1994 (link)). The PCR
products were purified using a QIA quick PCR kit (QIAGEN, Hilden, Germany) and
sequencing was outsourced to Macrogen (Seoul, Korea). The base sequences were
then compared with the NCBI GenBank DB using BLAST analysis (
8
ChIP-seq protocol for RNAPII, DCL1, and SE
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ChIP was performed as described (Bowler et al., 2004 (link)) with IP buffer prepared as described (Kaufmann et al., 2010 (link)). Chromatin was sonicated at 4°C with a Diagenode Bioruptor Pico for ∼15 min (30 s on/30 s off) to obtain 250–500-bp DNA fragments. Antibodies against total RNAPII (Abcam ab817, 5 µg/IP), DCL1 (Agrisera AS19 4307, 10 µg/IP), and SE (Agrisera AS09 532A, 10 µg/IP) were used with Dynabeads Protein G (Thermo Fisher Scientific). For decrosslinking and DNA isolation, samples were treated with Proteinase K (Thermo Fisher Scientific) for 6 h at 55°C followed by purification with a Qiaquick PCR Kit (Qiagen). Libraries were prepared using a MicroPlex Library Preparation Kit (Diagenode) and sequenced on the NextSeq platform.
9
Rapid Bacterial Identification by API 50 CHL and 16S rDNA
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The API 50 CHL kit (Biomerieux, La Balme-les-Grottes, France) was used to measure
sugar utilization and rapid identification of the isolated bacterial strains.
The API 50 CHL kit was inoculated with cultured colonies on MRS agar, and the
variation of color (yellow) in each was measured after culturing for 24 and 48 h
at 37°C, respectively. The results were used to identify bacterial
strains using Biomerieux DB (https://apiweb.biomerieux.com ).
In addition, genetic identification was performed by analyzing 16s rDNA. After
extracting genomic DNA using a genomic DNA preparation kit (Promega, Madison,
WI, USA), PCR was performed with the universal primers 27F (5’-AGA GTT
TGA TCC TGG CTC AG-3’) and 1492R (5’-TAC GGY TAC CTT GTT ACG ACT
T-3’) to amplify the 16s rDNA gene (Lim
et al., 2018 ). The PCR products were purified using the QIA quick PCR
kit (QIAGEN, USA), nucleotide sequencing was outsourced to Macrogen (Seoul,
Korea), and the sequences were compared with a DB using BLAST at GenBank on the
NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi ).
sugar utilization and rapid identification of the isolated bacterial strains.
The API 50 CHL kit was inoculated with cultured colonies on MRS agar, and the
variation of color (yellow) in each was measured after culturing for 24 and 48 h
at 37°C, respectively. The results were used to identify bacterial
strains using Biomerieux DB (
In addition, genetic identification was performed by analyzing 16s rDNA. After
extracting genomic DNA using a genomic DNA preparation kit (Promega, Madison,
WI, USA), PCR was performed with the universal primers 27F (5’-AGA GTT
TGA TCC TGG CTC AG-3’) and 1492R (5’-TAC GGY TAC CTT GTT ACG ACT
T-3’) to amplify the 16s rDNA gene (
et al., 2018
kit (QIAGEN, USA), nucleotide sequencing was outsourced to Macrogen (Seoul,
Korea), and the sequences were compared with a DB using BLAST at GenBank on the
NCBI website (
10
Cloning and Sequencing of Tumor DNA
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