DNA was extracted from peripheral blood using blood mini kit (PrimePrep Genomic DNA Isolation Kit, Genet Bio Inc.). For analysis of GSTT1 and GSTM1 genotypes of the subjects, polymerase chain reaction (PCR) amplification was performed using the following primers: GSTT1 forward primer 5′-TTC CTT ACT GGT CCT CAC ATC TC-3′; and reverse primer 5′-TCA CCG GAT CAT GGC CAG CA-3′; GSTM1 forward primer 5′ AGA CAG AAG AGG AGA AGA TTC 3′; and reverse primer 5′ TCC AAG TAC TTT GGC TTC AGT 3′. The PCR reaction was done as previously described.[18 (link)]
Primeprep genomic dna isolation kit
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The PrimePrep Genomic DNA Isolation Kit is a laboratory tool designed to extract and purify genomic DNA from a variety of biological samples. The kit utilizes a reliable protocol and proprietary reagents to efficiently isolate high-quality DNA, which can be used in downstream molecular biology applications.
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14 protocols using primeprep genomic dna isolation kit
1
Genotyping GSTT1 and GSTM1 via PCR
2
Case-Control Study of Colorectal Cancer
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This case–control study conducted on 60 patients (33 men and 27 women), [Table 1 ] and 74 healthy participants (30 men and 44 women), [Table 1 ]. Participants with no histologically confirmed CRC and no familial history of related cancers were randomly selected from the colonoscopy units of Al-Zahra hospital. Controls were individuals with no evidence of colonoscopy signs of CRC. They were recruited from the same residential areas. Informed consent was obtained from all the participants approved by Isfahan University of Medical Sciences Ethics Committee. The participants were interviewed and data on gender, age, smoking status, nonsteroidal anti-inflammatory drug (NSAID) usage, and physical activity were obtained using a structured questionnaire. Genomic DNA was extracted from 5-ml ethylenediaminetetraacetic acid-anticoagulated peripheral blood samples obtained from the participants by Prime Prep Genomic DNA Isolation Kit (GeNetBio, Korea). The quality and quantity of the extracted DNA was determined by agarose gel electrophoresis and spectrophotometer.
3
PBMC Isolation and DNA Extraction
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Following the established standard procedure,[19 ] PBMCs were isolated using density gradient centrifugation with Ficoll-Hypaque (Sigma, St. Louis, MO, USA) from the collected blood samples. Genomic DNA was then extracted from the PBMCs using the Prime Prep Genomic DNA Isolation Kit (GeNetBio, Korea). The quality and quantity of the extracted DNA were assessed through agarose gel electrophoresis and a NanoDrop Spectrophotometer, measuring the absorbance ratio at 260 nm.
4
Quantifying DNA Content in WJ Extracts
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To determine the total DNA content of native WJ and its decellularized extracts, 1 mg of each lyophilized sample was digested using 200 µL proteinase K (Invitrogen, USA) for 2–3 h. Total genomic DNA of native WJ and its decellularized extracts (n = 3) were isolated by PrimePrep Genomic DNA Isolation Kit (Genet Bio, South Korea) following the manufacturer’s instructions. The total amount of DNA was quantified by spectrophotometry (Nano Drop ND-0910, Thermo Scientific, USA). The ratio of optical densities of 260/280 nm indicated the purity and yield of nucleic acids, and absorbance in 260 nm showed the DNA quantity. The analysis was performed on three different donor samples of WJ.
5
Leishmania major Infection Model
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L. major strain (MRHO/IR/74/ER) used in this study was obtained from the spleen of L. major infected BALB/C mouse by culturing in NNN medium and then sub-culturing in RPMI 1640 supplemented with 12% heat -inactivated fetal bovine serum (FBS) and 1% Penstrep.
Female BALB/C mice (6–8 weeks old) were obtained from the Pasteur Institute of Iran. The animal care and the experimental protocols were approved by the Institutional Animal Care and Research Advisory Committee of the Shahid Beheshti University of Medical Sciences. Mice were infected intra-dermally at the base of their tail with 2 × 105L. major promastigotes in the stationary phase of growth. Eight weeks post infection, inguinal lymph nodes were isolated, placed into a 70 μm cell strainer (BD Falcon, Mexico) which was put into a sterile Petri dish containing RPMI 1640 and mashed gently using the plunger end of the syringe. Then, the cell suspension was collected and centrifuged. After centrifugation, cells were counted using a hemocytometer and a cell suspension containing 4 × 106 cells was stored at −70 °C until DNA extraction. DNA extraction was performed using a Spin column-based nucleic acid purification kit (Prime Prep Genomic DNA Isolation Kit, GeNet Bio, Daejeon, Korea) according to the manufacturer’s protocol. Extracted DNA was used as the template for real- time PCR.
Female BALB/C mice (6–8 weeks old) were obtained from the Pasteur Institute of Iran. The animal care and the experimental protocols were approved by the Institutional Animal Care and Research Advisory Committee of the Shahid Beheshti University of Medical Sciences. Mice were infected intra-dermally at the base of their tail with 2 × 105L. major promastigotes in the stationary phase of growth. Eight weeks post infection, inguinal lymph nodes were isolated, placed into a 70 μm cell strainer (BD Falcon, Mexico) which was put into a sterile Petri dish containing RPMI 1640 and mashed gently using the plunger end of the syringe. Then, the cell suspension was collected and centrifuged. After centrifugation, cells were counted using a hemocytometer and a cell suspension containing 4 × 106 cells was stored at −70 °C until DNA extraction. DNA extraction was performed using a Spin column-based nucleic acid purification kit (Prime Prep Genomic DNA Isolation Kit, GeNet Bio, Daejeon, Korea) according to the manufacturer’s protocol. Extracted DNA was used as the template for real- time PCR.
6
Yucatan Miniature Pig Ear Tissue DNA Extraction
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Ear tissue samples from 114 Yucatan miniature pigs were obtained from Optifarm solution Inc. (Ochang, Korea). Genomic DNAs were extracted using PrimePrep genomic DNA isolation Kit (GeNetbio, Daejeon, Korea). The concentration of the genomic DNA was measured using NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA) at a wavelength of 260 nm and stored at −20°C until use.
7
Identification of Pseudomonas aeruginosa Efflux Genes
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P. aeruginosa genomic DNA was extracted using the PrimePrep Genomic DNA isolation kit (GENETBIO). The efflux pump genes were determined by PCR using specific primers shown in table 1 . The PCR reaction was conducted in a final volume of 25 μl; and also, the following parameters were taken into account in this reaction: PCR buffer (10×) 2.5 μl, MgCl2 (50 mM) 0.75 μl, dNTPs (10 mM) 1 μl, forward and reverse primers (10 pmol/μl) 1 μl+ 1 μl, Taq DNA polymerase (5 U/μl) 1 μl, distilled water 16.75 μl and template DNA 1 μl. The PCR products were analyzed using electrophoresis (100 v, 45 min) in gels composed of 1.5% agarose stained with DNA staining dye and visualized under specific UV light.
8
Quantitative DNA Methylation Analysis by MethyQESD
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According to a previously described standard protocol,[23 ] the PBMCs were obtained by density gradient centrifugation over Ficoll-Hypaque (Ficoll-Hypaque, Sigma) from the peripheral blood samples of all individuals. DNA was extracted from the PBMCs using a Prime Prep Genomic DNA Isolation Kit (GeNetBio, Korea), and its quantity and quality were evaluated by gel electrophoresis and using a NanoDrop spectrophotometer, respectively.
Quantitative methylation analysis was accomplished using the MethyQESD technique.[24 (link)] This method is based on a combination of methylation-sensitive restriction enzymes and a real-time polymerase chain reaction (RT-PCR). According to the protocol, two different batches were considered, one a methylation-specific quantification digestion (MQD) with Hin6I and the other a methylation-sensitive endonuclease calibrator digestion (CalD) with the methylation-independent endonucleases, namely, XBaI and DraI. The sequence of primers was as follows: forward: 5’-TACCGCGCGTGGAGGAGACA-3’; reverse: 5’- GTGGGCAGGTACCGCAGCC-3’. The enzyme digestion and RT-PCR protocol were described in detail by Duppel et al.[25 (link)]
Quantitative methylation analysis was accomplished using the MethyQESD technique.[24 (link)] This method is based on a combination of methylation-sensitive restriction enzymes and a real-time polymerase chain reaction (RT-PCR). According to the protocol, two different batches were considered, one a methylation-specific quantification digestion (MQD) with Hin6I and the other a methylation-sensitive endonuclease calibrator digestion (CalD) with the methylation-independent endonucleases, namely, XBaI and DraI. The sequence of primers was as follows: forward: 5’-TACCGCGCGTGGAGGAGACA-3’; reverse: 5’- GTGGGCAGGTACCGCAGCC-3’. The enzyme digestion and RT-PCR protocol were described in detail by Duppel et al.[25 (link)]
9
Avian Haemosporidian Parasite Detection
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Blood and tissue genomic DNA were extracted using PrimePrep Genomic DNA Isolation Kit (GENETBIO Inc. Daejeon, South Korea) according to the instructions provided by manufacturer. DNA extraction was carried out using 50–100 μL of blood and 10 mg of tissue samples. The quality of the extracted DNA was checked by spectrophotometer (DeNovix Inc. USA). For detection of avian haemosporidian parasites, the ~480 base pair fragments of mt-DNA cytb were amplified. Nested PCR targeting was conducted using the primers of HaemNFl and HaemNR3 in the first step, and the primer pairs of HaemF, HaemR2 and HaemFL and HaemR2L in the second cycle to amplify Haemoproteus, Plasmodium and Leucocytozoon lineages (Bensch et al., 2000 ; Hellgren et al., 2004 (link)). The final volume of PCR reactions was 25 μl, comprising 50–100 ng/μl of total genomic DNA, 0.6 mM of each primer, 12.5 μl AMPLIQON red PCR master mix (AMPLIQON, Denmark), ddH2O (free nuclease, up to 25 μl). Besides, the ultrapure water and positive PCR product of previous experiments were used as negative and positive controls, respectively. All PCR products were visualized on 1% agarose gels, and the final positive amplicons were purified and sequenced using oligonucleotides of HaemF and HaemR2 (Haemoproteus and Plasmodium), and HaemFL and HaemR2L (Leucocytozoon) by BIONEER Inc. (Seoul, South Korea).
10
PBMC Isolation and Genomic DNA Extraction
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PBMCs were isolated via Ficol gradient density (Ficoll-Hypaque, Sigma) based on the standard protocol [27] . Genomic DNA was isolated from PBMCs by PrimePrep Genomic DNA Isolation Kit (GeNetBio, Korea).
The yield, purity, and suitability of DNA for the MethyQESD (methylation-quanti cation of endonucleaseresistant DNA) method was evaluated by spectrophotometry and agarose gel electrophoresis.
The yield, purity, and suitability of DNA for the MethyQESD (methylation-quanti cation of endonucleaseresistant DNA) method was evaluated by spectrophotometry and agarose gel electrophoresis.
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