29 human samples were prepared using eccDNA enrichment described previously (Møller et al., 2018 (link)) with column chromatography on an ion exchange membrane column (Plasmid Mini AX; A&A Biotechnology) and with precipitated DNA dissolved in 50 μl water for 7–21 ng total DNA (HS1 – HS12), 15–54 ng (HS20-HS23, HS25-HS27, HS29) and 23–54 ng (HS30-HS35, HS37-HS39) by Qubit dsDNA High Sensitivity assay. Furthermore, DNA from HS50, HS54, HS68, HS72 and HS73 ICI and IUI was also purified with the Plasmid Mini AX; A&A Biotechnology. In the case of HS50, HS54 and HS68, total DNA was also purified with the MagAttract HMW DNA Kit, 67563, QIAGEN according to manufacturer’s instruction, to investigate if the DNA extraction method affected the purification of eccDNA.
Plasmid mini ax
Manufactured by A&A Biotechnology
The Plasmid Mini AX is a laboratory equipment designed for the purification of plasmid DNA from bacterial cultures. It is a compact and efficient system that utilizes a modified alkaline lysis method to extract and purify plasmid DNA from small-scale cultures.
Automatically generated - may contain errors
7 protocols using plasmid mini ax
1
Eccentric DNA Enrichment and Purification
2
Sperm DNA Extraction and Quantification
3
Plasmid DNA Isolation and Analysis
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Plasmid DNA was isolated using Plasmid Mini AX (A&A Biotechnology) kit following manufacturer’s instruction. Plasmids were separated in a 0.7 % agarose gel, and molecular size of the bands was determined with GelCompar II 3.5; E. coli V-517 plasmids were used as molecular size markers. All plasmids were cut off the gel and eluted by Gel-Out kit (A&A Biotechnology). Integrons in plasmids were detected as described above.
4
Purification and Sequencing of Extrachromosomal DNA
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UACC-1598–4 cell line was alkaline treated to separate chromosomal DNA, lipids, and protein from eccDNA by rapid DNA denaturing–renaturing, followed by column chromatography on an ion exchange membrane column (Plasmid Mini AX; A&A Biotechnology), The eccDNA was purified according to the purification method provided in the document, linear and mitochondrial DNA were removed by endonucleases and rolling-circle amplification of eccDNA by Phi29 polymerase reactions (REPLI-g Midi Kit) amplifying DNA at 30 °C for 2 days for Circle-Seq and then the eccDNA was sequenced based on Illumina platform [17 (link)].
5
Plasmid DNA Isolation and Integrase Detection
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Plasmid DNA was isolated with Plasmid Mini AX (A&A Biotechnology) kit and separated in 0.7 % agarose gel with E. coli V-517 plasmids used as molecular size markers. The DNA was transferred to Immobilon-NY+ membrane (Millipore) and fixed by UV cross-linking. Class 1 integrase gene was detected by digoxigenin-labeled intI1 probes with the use of DIG DNA Labeling and Detection Kit (Roche) according to manufacturer’s instruction.
6
Isolation of Extrachromosomal DNA
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Samples were alkaline treated to separate chromosomal DNA, lipids, and protein from eccDNAs by rapid DNA denaturing–renaturing, followed by column chromatography on an ion exchange membrane column (Plasmid Mini AX; A&A Biotechnology). DNA precipitation was achieved by 45-min incubation at -20 °C (after column elution) and extended centrifugation at 9,788× g for 30 min at 2 °C. Precipitated DNA was dissolved in 50 µl water for total DNA 40–419 ng (tissue, T1–T16), 10–224 ng (blood, B1–B16), 22–33 ng (B6A–D) and 36–53 ng (B14A–D) by Qubit dsDNA High Sensitivity assay.
7
Multiplex Plasmid Quantification Protocol
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In brief, samples of 6 mg tissue (106 ± 3 × 105 genomes) or leukocyte pellets (104 ± 7 × 103 genomes) were resuspended in 0.63 mL L1 solution (Plasmid Mini AX; A&A Biotechnology) and supplemented with 15 µl Proteinase K (>0.1 U/µl, Life Technologies) before incubation overnight at 50 °C with agitation at 700 rpm (Eppendorf Thermomixer). After cell lysis, a control mixture of plasmids of different sizes and concentrations was added to each sample: 100 copies pSH63, 100 copies pUC19_yEGFP3, 10,000 copies YGPM3k20_pGP564_chrV, 20,000 copies pBR322, and 50,000 copies pUG72. At this point, 30 µl was sampled to assess the input DNA concentration by qPCR.
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