Prior to sample processing, 4 DNA extraction methods (QIAamp DNA minikit, Promega Wizard genomic extraction kit, Mo Bio PowerLyzer Ultraclean DNA isolation kit, and guanidium thiocyanate-EDTA-Sarkosyl extraction) were evaluated. The method with the best results for equal lysis of Gram-negative and Gram-positive bacteria, highest DNA yields from low starting material, and lowest intrinsic bacterial contamination was determined to be the Mo Bio PowerLyzer Ultraclean kit. Bacterial DNA was extracted from the TIVAP chamber flush liquid according to the manufacturer’s instructions. Control (DNA-free water as the template) extraction samples were run with each extraction in order to identify any potential DNA traces introduced during the extraction procedure. Extracted genomic DNA was verified by Tris-acetate-EDTA-agarose gel electrophoresis (1%), stained with GelRed, and quantified by applying 2.5 μl directly to a NanoDrop ND-1000 spectrophotometer.
Qiaamp dna mini kit
Manufactured by Promega
Sourced in United States
The QIAamp DNA Mini Kit is a DNA extraction and purification kit. It utilizes a silica-membrane-based technology to isolate DNA from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Abi prism 3100 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Exosap it, by Thermo Fisher Scientific (1 mentions)
Gotaq flexi dna polymerase, by Promega (1 mentions)
Vector nti advance, by Thermo Fisher Scientific (1 mentions)
Gotaq green master mix, by Promega (1 mentions)
Zymoclean gel dna recovery kit, by Zymo Research (1 mentions)
5 protocols using qiaamp dna mini kit
1
Comprehensive DNA Extraction Evaluation
2
Validating Experimental PCV2b Infection
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PCV2b viral genomic DNA was isolated using QIAamp DNA Minikit and amplified using GoTaq Flexi DNA Polymerase (Promega). The PCR products were purified using ExoSAP-IT (USB Corporation) and sequenced using dye terminators and ABI PRISM 3100 Genetic Analyzer (Life Technologies). The assembled sequence (GenBank accession number: KP016747) was aligned to the publicly available PCV2 genome sequences using CLUSTALW2 [27 (link)].
Following the experimental challenge, viral genomic DNA was isolated from random, high and low viral load pigs (n = 18) representing most of the batches and sequenced to validate the genetics of PCV2b strain used for experimental infection.
Following the experimental challenge, viral genomic DNA was isolated from random, high and low viral load pigs (n = 18) representing most of the batches and sequenced to validate the genetics of PCV2b strain used for experimental infection.
3
Establishing Lentiviral CRISPR Knockout Clones
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The following gRNA sequences were cloned into the pNGx-LV-g003 lentiviral backbone (DeJesus et al., 2016 (link)) and transduced into the HCT116-Cas9 clone described above at an MOI 0.5: ATP2C1 5’- GAACTCTATCCCCAACAGAA-3’, FERMT2 5’-GGTGGGAAAAGAAGAGAACT-3’, DUSP5 5’- GCGCTACGTGCTGCCCGACG-3’. The cells were selected for 4 days using 2 μg/ml puromycin and transduction efficiency (RFP signal) was assessed by FACS analysis. Selected cells were diluted to a density of 100-300cells / 20 ml and seeded in 15 cm dishes in puromycin containing medium. After 2 weeks, single colonies were picked using cloning discs, expanded for another 10-15 days and frozen down as master stocks.
Editing was assessed by genotyping as follows. Genomic DNA was extracted for each clone using Qiagen’s QIAamp DNA mini Kit (51304) and the DNA regions across each sgRNA were amplified by PCR using Promega’s GoTaqGreen master mix (M7122) following the standard protocol. The PCR bands were extracted using Zymo Research’s Zymoclean Gel DNA Recovery Kit (D4001) and submitted to Sanger sequencing (Microsynth standard service). The sequences were analysed using the Vector NTI Advance (Invitrogen, version 11.5.4).
Editing was assessed by genotyping as follows. Genomic DNA was extracted for each clone using Qiagen’s QIAamp DNA mini Kit (51304) and the DNA regions across each sgRNA were amplified by PCR using Promega’s GoTaqGreen master mix (M7122) following the standard protocol. The PCR bands were extracted using Zymo Research’s Zymoclean Gel DNA Recovery Kit (D4001) and submitted to Sanger sequencing (Microsynth standard service). The sequences were analysed using the Vector NTI Advance (Invitrogen, version 11.5.4).
4
MRSA Detection in S. aureus Isolates
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S. aureus isolates were tested for MRSA using oxacillin resistance screen agar (ORSA) (Merck) [16 ]. ORSA was inoculated directly with an isolated colony of S. aureus prepared as a liquid suspension approximately equivalent to 0.5 McFarland turbidity standards. The medium was prepared according to the manufacturer’s instructions before inoculation. The inoculated plates were incubated for 18–24 h at 37°C. The colonies showing blue indicators were recorded as MRSA, and colonies with white on the agar were recorded as methicillin-susceptible S. aureus after 24 h of incubation. All the MRSA verified by the ORSA were tested using a polymerase chain reaction (PCR) to detect the presence of the mecA gene [17 ]. The DNA extraction process was performed according to the QIAamp DNA Mini Kit (Promega, USA) protocol (51304 and 51306) [17 ]. The PCR method and primers were used as described by Ramandinianto et al. [18 ], as shown in Table-1 [18 ]. Positive control was S. aureus ATCC BAA 1026, and negative control was S. aureus ATCC 25923.
5
Detecting ESBL-Producing E. coli via PCR
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E. coli isolates were studied for the presence of ESBL using the double-disk synergy test (DDST). The antibiotic disks used for DDST were amoxicillin-clavulanate (20/10 μg), cefotaxime (30 μg), and ceftazidime (30 μg) [19 ]. The ESBL-producing E. coli detected was further examined at a molecular level. Bacterial DNA was extracted using the QIAamp DNA Mini Kit (Promega) protocol according to Kristianingtyas et al. [19 ], and the blaTEM gene was detected using the PCR method as described by Putra et al. [20 ] and Ansharieta et al. [21 ] as indicated in Table-1 . After the amplification, products were visualized by exposure of the gel to ultraviolet light and subsequently photographed and documented using agel documentation system (Promega). Positive control was E. coli ATCC 35218, and negative control was E. coli ATCC 25922.
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