Transposon mutagenesis was carried out via biparental conjugations using E. coli SM10 λpir carrying plasmid pFAC as the donor strain (Wong and Mekalanos, 2000 (link)) and PAO1 as the recipient strain. After incubating PAO1 and E. coli cells on LB agar plates at 37°C for 6 h, bacteria were collected, re-suspended in PBS and then plated onto PIA supplemented with gentamicin (200 μg ml−1). Mucoid colonies were identified. The chromosomal DNA of mucoid mutants was isolated using the QIAamp genomic DNA Extraction kit (Qiagen, USA). Approximately, 2 μg of DNA was digested with SalI overnight at 37°C, followed by purification and self-ligation using Fast-Link DNA ligase (Epicentre, USA). The circular closed DNA was used as a template for inverse PCR using GM3OUT and GM5OUT primers (Qiu et al., 2008b (link)). The PCR products were purified and sequenced. Finally, Southern-blot hybridization was also conducted to monitor the copy number of transposon insertions using the Gmr gene as the probe (Head and Yu, 2004 (link)).
Qiaamp genomic dna extraction kit
Manufactured by Qiagen
Sourced in Germany, United States
The QIAamp genomic DNA extraction kit is a laboratory equipment product designed for the isolation and purification of genomic DNA from a variety of biological samples. It utilizes a simple silica-based membrane technology to effectively capture and purify DNA, making it suitable for a range of downstream applications.
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8 protocols using qiaamp genomic dna extraction kit
1
Transposon Mutagenesis in Pseudomonas aeruginosa
2
Quantifying Transplanted hADMSCs in Mice Livers
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To quantify the transplanted hADMSCs that homed to the mice livers, a human Down syndrome sequence-based real-time PCR quantification system was used in the current study [7 (link)]. In brief, hepatic genomic DNA samples were extracted using a QIAamp genomic DNA extraction kit (Qiagen, Hilden, Germany). A pair of primers (5′- ATGCTGATGTCTGGGTAGGGTG-3′ and 5’-TGAGTCAGGAGCCAGCGTATG-3′) that generate a 141-bp fragment of human Down syndrome region at chromosome 21 was used to quantify the human-derived cells. To verify the quantitative PCR results, we performed an immunohistochemistry (IHC) assay using human albumin antibody in sectioned mice liver samples.
3
Genomic DNA Extraction from Blood Samples
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DNA was extracted from peripheral blood samples of the affected family using the manufacturer’s protocol using QIAamp genomic DNA extraction kit (QIAGEN, Germantown, MD, USA). DNA quantification and quality assessment was done by NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis was done to confirm the integrity of genomic DNA before sequencing [26 (link),27 (link)].
4
Quantifying Human Cells in Mouse Liver
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To quantify the transplanted hUCMSCs and i-Heps that homed at the mice liver, a recently established real-time PCR quantification system has been used in the current study21 (link). Briefly, genomic DNA at day 7 post-treatment was extracted from mouse livers using QIAamp genomic DNA extraction kit (Qiagen, Hilden, Germany). A pair of primers (see Supplementary Table 1 for sequence information) that generate a 141-bp fragment of human Down syndrome region at chromosome 21 were used to quantify the human-derived cells. The real-time PCR reaction was performed using an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA) for 40 cycles with denaturing at 95 °C for 30 seconds and annealing at 63 °C for 34 seconds, with a SYBR-Green Realtime PCR mix (Takara, Dalian, China).
5
Quantifying Bacterial Populations via qPCR
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To quantify each bacterial group, DNA from the respective reference strain was used for construction of a standard curve. The bacterial strains, efficiency, and coefficient of determination (R2 value) of the standard curve for qPCR are listed in Table 3 . Culture conditions used to grow the standard bacterial strains are described in Supplementary Table 1 . qPCR was performed as described above with serially diluted bacterial genomic DNA extracted from the reference strains using the QIAamp genomic DNA extraction kit (Qiagen), and the respective copy numbers of 16S rRNA were calculated. Each standard curve was constructed by plotting CT values against bacterial quantity (in 16S rRNA).
6
Transfection and Reinfection of MT-4 and Jurkat Cells
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For transfection, 2.5 × 106 MT-4 cells and Jurkat cells were mixed with 1 μg and 2.5 μg plasmid DNA, respectively, in 500 μl of 0.8 mg/ml DEAE-dextran solution. After 15 min of incubation at 37°C, cells were washed with 4 ml of 1× STBS (25 mM Tris-HCl [pH 7.4], 0.6 mM Na2HPO4, 5 mM KCl, 140 mM NaCl, 0.7 mM CaCl2, 0.5 mM MgCl2) and pelleted by centrifugation. The cell pellets were resuspended in 2.5 ml RPMI medium and transferred to tissue culture flasks. Aliquots of supernatants were collected to monitor RT activity, and cells were split every other day. For reinfection, 1.5 × 106 MT-4 cells or 2.5 × 106 Jurkat cells were mixed with RT-normalized viral stocks, incubated for 2 h at 37°C, and pelleted by centrifugation. Cell pellets were resuspended in 2.5 ml RPMI medium and transferred to tissue culture flasks. On the days of peak RT activity, the genomic DNA was extracted using a QIAamp genomic DNA extraction kit (Qiagen), and CA-coding regions were amplified and sequenced (Macrogen).
7
Mycobacterial DNA Extraction and Identification
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The mycobacterial DNA was extracted using QIAamp® genomic DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The quality and quantity of the extracted DNA were evaluated using Nanodrop (DeNovix Inc., Wilmington, DE, USA). Extracted DNA was stored at –20°C for later analysis. In this study, an IS6110-based PCR assay was used to confirm M. tuberculosis strains as previously described [14 (link)].
8
Quantifying Transplanted Cells in Liver
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To quantify the transplanted hUCMSCs and i-Heps that homed at the mice liver, a recently established real-time PCR quantification system has been used in the current study [18] (link). Briefly, genomic DNA was extracted from mouse livers using QIAamp genomic DNA extraction kit (Qiagen, Hilden, Germany). A pair of primers (forward: 5′-ATGCTGATGTCTGGGTAGGGTG-3′ , reverse: 5′-TGAGTCAGGAGCCAGCGTATG-3′ ) that generate a 141-bp fragment of human Down syndrome region at chromosome 21 were used to quantify the human-derived cells. The real-time PCR reaction was performed using an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA) for 40 cycles with denaturing at 95°C for 30 seconds and annealing at 63°C for 34 seconds, with a SYBR-Green Realtime PCR mix (Takara, Dalian, China).
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