Approximately 25 mg of tissue specimens collected from the wild rodents were ground using a cell strainer (70 μm; Falcon, Corning, NY, USA) and 180 μL of ATL buffer from the QIAamp DNA Blood and Tissue Mini Kit (Qiagen, Hilden, Germany). The tissue suspension was treated with 20 µL of proteinase K and incubated overnight at 56 °C for complete tissue lysis. Genomic DNA was extracted using the QIAamp DNA Blood and Tissue Mini Kit (Qiagen) according to the manufacturer’s instructions.
Qiaamp dna blood and tissue mini kit
Manufactured by Qiagen
Sourced in Germany
The QIAamp® DNA Blood and Tissue Mini Kit is a laboratory equipment product designed for the purification of DNA from various biological samples, including blood, tissue, and cells. It utilizes a spin-column based method to efficiently extract and purify DNA. The kit includes necessary buffers, reagents, and collection tubes to facilitate the DNA extraction process.
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Lab products found in correlation
Nanodrop nd 1000, by Thermo Fisher Scientific (5 mentions)
Nd 1000, by Thermo Fisher Scientific (1 mentions)
Prism 5.0 statistical, by GraphPad (1 mentions)
Cell strainer, by BD (1 mentions)
Nd 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Gentra puregene tissue dna extraction kit, by Qiagen (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
11 protocols using qiaamp dna blood and tissue mini kit
1
Genomic DNA Extraction from Wild Rodents
2
DNA Extraction from Whole Blood and Bone Marrow
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After thawing at room temperature, 200 μl of whole blood and bone marrow were subjected to DNA extraction using a commercial kit (QIAamp™ DNA Mini Kit Blood and Tissue, Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. Negative control purifications using ultrapure water were performed in parallel to monitor cross-contamination in each batch of 30 samples. The concentration and purity of extracted DNA were assessed by spectrophotometry (ND-1000, Nano-Drop Technologies, Wilmington, DE, USA) by measuring the absorbance at 260 and 280 nm, respectively. Thereafter, DNA aliquots were stored at -20 °C until molecular testing.
3
DNA Extraction from Dried Blood Spots
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DNA was extracted from filter paper using QIAamp™ DNA Mini kit blood and tissue (QIAGEN Germany) according to the manufacturers’ instructions. Briefly, three punched out circles from the dried blood spots were placed into a 1.5 ml microcentrifuge tube and 180 µl ATL (tissue lysing buffer) was added and incubated at 85°C for 10 min. Twenty microliters of proteinase K was added to the mix and incubated for 1hr at 56°C. 200 µl of ATL buffers (tissue lysing buffer) was added to the sample and was incubated for another 10 min at 70°C. Two hundred microliter of 96%–100% ethanol was added to the sample, vortexed thoroughly and then transferred to the QIAamp Mini spin column. The sample was centrifuged at 8000rpm for 1 min and the flow through (filtrate) discarded following which 500 µl of wash buffers (AW1 and AW2) were added to the mini spin column and then centrifuged for 1 min at 8000rpm for AW1 and 14000 rpm for AW2 for 3 min. Finally, 150 µl of Buffer AE (Elution buffer) was added to the spin column, incubated for 1 min at 25°C and centrifuged at 8000rpm for 1 min to obtain the final yield of the DNA. The extracted DNA was stored at –20°C until use.
4
Quantification of HSV DNA in NHP Samples
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NHP samples for qPCR analysis (25 mg) were placed in sterile microcentrifuge tubes in proteinase K containing digestion buffer and digested overnight at 56°C. DNA was extracted into 200 μL elution buffer from NHP samples using the QIAamp DNA Blood and Tissue Mini Kit (QIAGEN) per the manufacturer’s instructions. The tissue preparation, DNA extraction, and TaqMan preparation steps were performed in a laminar flow hood in a laboratory that does not work with HSVs. Extracted DNA samples were incubated with the following HSV-specific primers and probes for HSV polymerase (sequences kindly provided by Dr. Fred Lakeman): PolF (forward), 5′-ACC GCC GAA CTG AGC AGA C-3′; and PolR (reverse), 5′-TGA GCT TGT AAT ACA CCG TCA GGT-3′. The fluorescent-labeled probe sequence was 5′-6FAM-CGC GTA CAC CAA CAA GCG CCT G-TAMRA-3′. HSV GEs of the amplified product were measured from triplicate samples using an ABI 7300 TaqMan machine (Applied Biosystems ) and compared against logarithmic dilutions of a positive control DNA sequence (106 – 101 copies). Descriptive statistical analyses (mean and SD) were used to compare differences in DNA copy numbers between samples using Prism 5.0 statistical software (GraphPad).
5
DNA Extraction and Quantification
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DNA quantification assay was performed using a QIAamp® DNA Blood and Tissue Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions, and the DNA yield (ng/μL) was quantified spectrophotometrically (the optical density (OD) at γ = 260 nm), using the NanoDrop® ND-1000 (Nanodrop Technologies Inc., Wilmington, DE, USA).
6
Quantifying Residual DNA in Decellularized Scaffolds
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DNA count assay was performed to investigate residual nuclear material of decellularized scaffold by QIAamp® DNA Blood and Tissue Mini Kit (QiagenGmbH, Hilden, Germany) according to the instructions. The extracted DNA materials (ng/μL) were analyzed by spectrophotometer (the optical density (OD) at γ = 260 nm) using the NanoDrop® ND-1000 (Nanodrop Technologies Inc., Wilmington, DE, USA).
7
Comprehensive Tissue Analysis After Decellularization
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Hematoxylin & Eosin and Hoechst staining were performed to evaluate the presence of nuclei in the tissues. To quantify DNA content, QIAamp® DNA Blood and Tissue Mini Kit (Qiagen GmbH, Hilden, Germany) was used. According to the manufacturer's instruction, 25 mg of lyophilized decellularized and intact bones was incubated in proteinase K at 56°C until it was completely lysed. The samples were transferred to a microcentrifuge tube and washed by a buffer. DNA was eluted by adding ethanol and centrifuged in a mini spin column and extracted DNA quanti ed by spectrophotometer at γ = 260 nm, using the NanoDrop® ND-1000 (Nanodrop Technologies Inc., Wilmington, DE, USA).
Histochemical staining was done to evaluate the retention of ECM content after decellularization. Alcian blue (pH 2.5), and PAS staining were performed to detect GAGs and neutral sugars, respectively. Collagen and elastic ber retention was con rmed by Masson's trichrome and aldehyde fuchsin staining.
Histochemical staining was done to evaluate the retention of ECM content after decellularization. Alcian blue (pH 2.5), and PAS staining were performed to detect GAGs and neutral sugars, respectively. Collagen and elastic ber retention was con rmed by Masson's trichrome and aldehyde fuchsin staining.
8
Evaluating Decellularized Bone Composition
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Hematoxylin & Eosin and Hoechst staining were performed to evaluate the presence of nuclei in the tissues. To quantify DNA content, QIAamp® DNA Blood and Tissue Mini Kit (Qiagen GmbH, Hilden, Germany) was used.
According to the manufacturer's instruction, 25 mg of lyophilized decellularized and intact bones was incubated in proteinase K at 56 °C until it was completely lysed. The samples were transferred to a microcentrifuge tube and washed by a buffer. DNA was eluted by adding ethanol and centrifuged in a mini spin column and extracted DNA quantified by spectrophotometer at γ = 260 nm, using the NanoDrop® ND-1000 (Nanodrop Technologies Inc., Wilmington, DE, USA).
Histochemical staining was done to evaluate the retention of ECM content after decellularization. Alcian blue (pH 2.5), and PAS staining were performed to detect GAGs and neutral sugars, respectively. Collagen and elastic fiber retention was confirmed by Masson's trichrome and aldehyde fuchsin staining.
According to the manufacturer's instruction, 25 mg of lyophilized decellularized and intact bones was incubated in proteinase K at 56 °C until it was completely lysed. The samples were transferred to a microcentrifuge tube and washed by a buffer. DNA was eluted by adding ethanol and centrifuged in a mini spin column and extracted DNA quantified by spectrophotometer at γ = 260 nm, using the NanoDrop® ND-1000 (Nanodrop Technologies Inc., Wilmington, DE, USA).
Histochemical staining was done to evaluate the retention of ECM content after decellularization. Alcian blue (pH 2.5), and PAS staining were performed to detect GAGs and neutral sugars, respectively. Collagen and elastic fiber retention was confirmed by Masson's trichrome and aldehyde fuchsin staining.
9
Decellularized Bone Tissue Analysis
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Hematoxylin & Eosin and Hoechst staining were performed to evaluate the presence of nuclei in the tissues. To quantify DNA content, QIAamp® DNA Blood and Tissue Mini Kit (Qiagen GmbH, Hilden, Germany) was used. According to the manufacturer's instruction, 25 mg of lyophilized decellularized and intact bones was incubated in proteinase K at 56 °C until it was completely lysed. The samples were transferred to a microcentrifuge tube and washed by a buffer. DNA was eluted by adding ethanol and centrifuged in a mini spin column and extracted DNA quanti ed by spectrophotometer at γ = 260 nm, using the NanoDrop® ND-1000 (Nanodrop Technologies Inc., Wilmington, DE, USA).
Histochemical staining was done to evaluate the retention of ECM content after decellularization. Alcian blue (pH 2.5), and PAS staining were performed to detect GAGs and neutral sugars, respectively. Collagen and elastic ber retention was con rmed by Masson's trichrome and aldehyde fuchsin staining.
Histochemical staining was done to evaluate the retention of ECM content after decellularization. Alcian blue (pH 2.5), and PAS staining were performed to detect GAGs and neutral sugars, respectively. Collagen and elastic ber retention was con rmed by Masson's trichrome and aldehyde fuchsin staining.
10
Optimized DNA Extraction from Blood, Tissue, and Sand Flies
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DNA was extracted from blood (200 μl) and spleen (25mg) by QIAamp Blood and tissue DNA mini kit (Qiagen, Hilden, Germany) in 60 μl of nuclease free water (Milli-Q) in accordance with the manufacturer’s instructions. From single blood-fed sand flies, DNA was extracted 48 hrs. after xenodiagnosis via Gentra Puregene Tissue DNA Extraction Kit (Qiagen). To achieve maximum yield, tissue digestion was performed overnight with proteinase K in lysis buffer and increased the protein precipitation timing at -20°C for overnight. DNA was eluted in 30 μl of Milli-Q and stored at -20°C for downstream processing. This protocol was optimized for individual sand flies [46 (link)]. The quality of extracted DNA from samples (Blood, tissue and sand flies) was assessed by spectrophotometer (ND-2000 spectrophotometer; Thermo Scientific, USA). DNA samples having 260/280 ratio in range 1.8–2.0 with 260/230 ratio above 1.5 were used for qPCR experiments, which included approximately 95% of samples.
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