DNA Extraction: Three ml of blood was collected from each subject in a sterile EDTA vacutainer. Samples were stored at -20°C till DNA extraction. Genomic DNA was extracted using the QIAamp DNA Mini isolation kit (QIAGEN, # 51304) following the manufacturer’s instructions and was stored at -20°C until the analysis. The DNA concentration was determined at a 260 / 280 nm absorbance ratio by NanoDrop 2000c Spectrophotometer (Thermo Fisher).
Qiaamp dna mini isolation kit
Manufactured by Qiagen
Sourced in Germany
The QIAamp DNA Mini isolation kit is a DNA purification system designed for the extraction and purification of DNA from a variety of sample types. It utilizes a silica-membrane-based technology to efficiently bind and purify DNA, making it suitable for use in various applications that require high-quality genomic DNA.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Ethidium bromide, by Thermo Fisher Scientific (2 mentions)
Chemidoc, by Bio-Rad (2 mentions)
Steponeplus real time pcr detection system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Ae buffer, by Qiagen (1 mentions)
Taqman snp genotyping assay, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulphoxide, by Merck Group (1 mentions)
Rpmi 1640 with glutamax, by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
7 protocols using qiaamp dna mini isolation kit
1
Blood DNA Extraction Protocol
2
cffDNA Isolation and Sonication
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DNA isolation was performed using the QIAamp DNA Mini isolation kit (Qiagen, Hilden, Germany) from isolated primary AECs following the manufacturer’s instructions. DNA was quantified using a NanoDrop (Thermo Fisher Scientific, Waltham, MA, United States). To create smaller fragments of cffDNA that would be more similar to those seen in AF, it was sonicated after dilution to 10 ng/μl in AE buffer (Qiagen, Hilden, Germany) for 4 min within an ultra sonicating water bath (PS-20A, Vevor, Shanghai, China). cffDNA fragment size of the whole (non-sonicated) and sonicated samples were verified on a 2% agarose gel with ethidium bromide (Invitrogen, Waltham, MA, United States) and visualized under UV transillumination using a Chemidoc (Bio-Rad, Hercules, CA, United States).
3
DNA Isolation and Sequencing Protocol
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Genomic DNA was isolated from tissue, using the QIAamp DNA mini isolation kit (Qiagen, Valencia, CA, USA), and the sequence of interest was amplified by PCR. Primers were described by Wu et al.[37 (link)]. PCR products were subsequently sequenced by the dideoxy chain-termination method, using the ABI PrismTM BigDye Terminator kit (Perkin Elmer, Foster City, CA, USA), run on the ABI Prism Genetic Analyser 3100 automatic DNA autosequencer (Perkin Elmer), and analysed with ABI sequence Alignment Editor software.
4
Genomic DNA Extraction from Bone Marrow
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Bone marrow samples (1ml) from patients and controls were collected on EDTA, bone marrow was treated with erythrocyte lysis solution; leukocytes were collected and stored in buffer RLT (1x107leukocytes) at -80 °C till use for complete DNA extraction.
Total genomic DNA was extracted from bone marrow cells of patients with AML and Control using QIAamp DNA Mini isolation kit (QIAGEN) (Cat No.: 51304) following standard procedures according to the manufacturer’s instructions.
Total genomic DNA was extracted from bone marrow cells of patients with AML and Control using QIAamp DNA Mini isolation kit (QIAGEN) (Cat No.: 51304) following standard procedures according to the manufacturer’s instructions.
5
Genotyping FCGR3A rs396991 in PBMCs
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Genomic DNA was extracted from 2 × 106 snapfrozen PBMC using the QIAamp DNA Mini isolation kit (Qiagen, Venlo, The Netherlands) according to manufacturer’s instruction. FCGR3A rs396991 genotype was determined by the StepOnePlus Real-Time PCR detection system (Applied Biosystems, Darmstadt, Germany) using Taqman SNP Genotyping assay (assay ID C_25815666_10; ThermoFisher Scientific Inc, Bleiswijk, The Netherlands) and Taqman Universal PCR Master Mix according to manufacturer’s instruction.
6
Extraction and Fragmentation of cffDNA
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DNA isolation was performed using the QIAamp DNA Mini isolation kit (Qiagen, Hilden, Germany) from isolated primary AECs following the manufacturer’s instructions. DNA was quantified using a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). To create smaller fragments of cffDNA that would be more like those seen in AF, it was sonicated as previously described [13 (link)]. Non-sonicated “whole DNA” was designated as LF cffDNA and 4-min sonicated DNA was designated as SF cffDNA. cffDNA fragment size of the LF cffDNA and SF cffDNA samples were verified on a 2% agarose gel with ethidium bromide (Invitrogen, Waltham, MA, USA) and visualized under UV transillumination using a Chemidoc (Bio-Rad, Hercules, CA, USA) as previously described [13 (link)].
7
FCGR3A Genotyping from Frozen PBMCs
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Prior to and at time of the for-cause biopsy, peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples by using Ficoll-Paque (GE HealthCare, Uppsala, Sweden). Two million PBMCs of the sample, obtained prior to transplantation, were snap-frozen in liquid nitrogen for isolation of genomic DNA (see below). The remaining PBMCs were washed, frozen at 10× 10 6 /vial in RPMI-1640 with Glutamax (GibcoBRL, ) supplemented with 100 IU/mL penicillin/streptomycin and 10% heatinactivated pooled serum and 10% dimethyl sulphoxide (Sigma Aldrich, Darmstadt, Germany) in liquid nitrogen until further use.
FCGR3A 158 F/V (rs396991) genotyping Genomic DNA was extracted from 2x10 6 snapfrozen PBMC using the QIAamp® DNA Mini isolation kit (Qiagen, Venlo, The Netherlands) according to manufacturer's instruction. FCGR3A rs396991 genotype was determined by the StepOnePlus Real-Time PCR detection system (Applied Biosystems, Darmstadt, Germany) using Taqman® SNP Genotyping assay (assay ID C_25815666_10; ThermoFisher Scienti c Inc, Bleiswijk, The Netherlands) and Taqman® Universal PCR Master Mix according to manufacturer's instruction.
FCGR3A 158 F/V (rs396991) genotyping Genomic DNA was extracted from 2x10 6 snapfrozen PBMC using the QIAamp® DNA Mini isolation kit (Qiagen, Venlo, The Netherlands) according to manufacturer's instruction. FCGR3A rs396991 genotype was determined by the StepOnePlus Real-Time PCR detection system (Applied Biosystems, Darmstadt, Germany) using Taqman® SNP Genotyping assay (assay ID C_25815666_10; ThermoFisher Scienti c Inc, Bleiswijk, The Netherlands) and Taqman® Universal PCR Master Mix according to manufacturer's instruction.
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