PCR using var-specific primers from Salanti et al. [26 (link)] with modifications [44 (link)] was carried out using the following conditions: 94 °C 3 min, 94 °C 30 s, 48 °C 45 s for 30× cycles, then 70 °C 30 s, 70 °C 3 min. PCR products were run on a 1% agarose gel, bands were cut out and purified with NucleoSpin® Extract Kit (Macherey–Nagel) according to the manufacturer’s protocol. Preparation of the DNA for sequencing was as follows: the reaction mix containing 1× sequencing buffer, 10% Big Dye Terminator v1.1 (Applied Biosystems), sequencing buffer (Applied Biosystems), 125 µM primer, 60% dH2O, 1–5 ng DNA were run with the following conditions: 94 °C 10 s, 50 °C 5 s, 25 cycles, 60 °C 4 min. All samples were purified using a 6.7% Sephadex (w/v) column. Sequences were aligned and a consensus sequence was generated with the help of BioEdit sequence alignment editor (http://www.mbio.ncsu.edu/BioEdit/bioedit.html ).
Sequencing buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Sequencing buffer is a specialized solution used in DNA sequencing procedures. It is designed to maintain the optimal pH and ionic conditions required for the DNA sequencing reaction to occur. The buffer helps to create a suitable environment for the enzymes and reagents involved in the sequencing process.
Automatically generated - may contain errors
Lab products found in correlation
3500xl genetic analyzer, by Thermo Fisher Scientific (2 mentions)
Bigdye v3, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v1, by Thermo Fisher Scientific (1 mentions)
Nucleospin extract kit, by Macherey-Nagel (1 mentions)
Sephadex g50 columns, by GE Healthcare (1 mentions)
Applied biosystems 3130 dna analyser, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator v3.1 ready reaction mix, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator cycle sequencing kit v3, by Thermo Fisher Scientific (1 mentions)
Forward and reverse primer, by Merck Group (1 mentions)
Dntps, by Thermo Fisher Scientific (1 mentions)
Bigdye xterminator purification kit, by Thermo Fisher Scientific (1 mentions)
Hotstartaq dna polymerase, by Qiagen (1 mentions)
Mgcl2, by Qiagen (1 mentions)
Bigdye terminator v3, by Thermo Fisher Scientific (1 mentions)
Exosap, by Thermo Fisher Scientific (1 mentions)
3730 dna analyzer, by Thermo Fisher Scientific (1 mentions)
Abi 3500 genetic analyser, by Thermo Fisher Scientific (1 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Transformaid bacterial transformation kit, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator, by Thermo Fisher Scientific (1 mentions)
9 protocols using sequencing buffer
1
PCR-based Sequencing of var Genes
2
Sanger Sequencing Protocol with BLAST Analysis
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Big-Dye v3.1 (Applied Biosystems, USA): 2 µL
Sequencing buffer (Applied Biosystems, USA): 3 µL
Primer 3.2 pmol/µL: 1 µL
ddH2O: 12 µL
For each reaction, 2 µL template was added to 18 µL master mix and the PCR thermocycler was programmed. Nucleotide sequencing was performed at the Sequencing core facility of the International Institute for Tropical Agriculture (IITA), Ibadan.
Searching for nucleotide sequence homology was performed using the Basic Local Alignment Search Tool (BLAST) available at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST ). Using the BLAST, the ultimate confirmation of gene sequences was made. First, an ExPasy convert to the amino acid sequence was done. All base and amino acid sequences were then used for a search in the database for homology of sequences. The BLAST-n and BLAST-p were used for nucleotide and amino-acid alignments, respectively.
Sequencing buffer (Applied Biosystems, USA): 3 µL
Primer 3.2 pmol/µL: 1 µL
ddH2O: 12 µL
For each reaction, 2 µL template was added to 18 µL master mix and the PCR thermocycler was programmed. Nucleotide sequencing was performed at the Sequencing core facility of the International Institute for Tropical Agriculture (IITA), Ibadan.
Searching for nucleotide sequence homology was performed using the Basic Local Alignment Search Tool (BLAST) available at the National Center for Biotechnology Information website (
3
DNA Sequencing Protocol using Big Dye
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Sequencing reactions were performed in both directions using a Big Dye Terminator cycle sequencing kit v3.1 (Applied Biosystems, Foster City, CA, USA), and the same set of PCR primers. Each sequencing reaction was carried in a total volume of 10 μL containing 1 μL of purified PCR product, 0.5 μL of Big Dye® Terminator v3.1 Ready Reaction Mix (PE Applied Biosystems), 1× sequencing buffer (PE Applied Biosystems), and 3.6 pmol of F or R primer, and the remaining volume consisted of ultrapure water. The sequencing products were purified with Sephadex G50 columns (GE Healthcare) and analyzed in an Applied Biosystems 3130 DNA Analyser (PE Applied Biosystems, Warrington, UK). Sequences were edited using the Sequencher software v. 5.2.4 (Genes Codes Corporation, Ann Arbor, MI, USA) and the primer regions were removed.
4
KRAS Mutation Analysis by PCR and Sequencing
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KRAS (codons 12 and 13) hotspot mutation regions were analysed by PCR, followed by direct sequencing, as previously described[38 (link)]. PCR was performed at a final volume of 15 μL under the following conditions: 1.5 μL of buffer (Qiagen, Venlo, The Netherlands), 2 mmol/L MgCl2 (Qiagen, Venlo, The Netherlands), 100 mmol/L dNTPs (Invitrogen, Carlsbad, Califórnia, United States), forward and reverse primers at 0.2 mmol/L (Sigma Aldrich), 1 unit of HotStarTaq DNA polymerase (Qiagen, Venlo, The Netherlands) and 1 μL of DNA at 50 ng/μL. The KRAS primers used were 5’-GTGTGACATGTTCTAATATAGTCA-3’ (forward) and 3’-GAATGGTCCTGCACCAGTAA-5’ (reverse)[39 (link)].
PCR products were purified with ExoSAP (GE Technology, IL, United States) and then added to a sequencing reaction mix containing 1 μL of BigDye (Applied Biosystems, Foster City, CA, United States), 1.5 μL of sequencing buffer (Applied Biosystems, Foster City, CA, United States) and 1 μL of primer, followed by post-sequencing purification with a BigDye XTerminator Purification Kit (Applied Biosystems, Foster City, CA, United States) according to the instructions from the manufacturer. Direct sequencing was performed on a 3500 xL Genetic Analyzer (Applied Biosystems, Foster City, CA, United States). All mutations were confirmed in a second, independent PCR experiment.
PCR products were purified with ExoSAP (GE Technology, IL, United States) and then added to a sequencing reaction mix containing 1 μL of BigDye (Applied Biosystems, Foster City, CA, United States), 1.5 μL of sequencing buffer (Applied Biosystems, Foster City, CA, United States) and 1 μL of primer, followed by post-sequencing purification with a BigDye XTerminator Purification Kit (Applied Biosystems, Foster City, CA, United States) according to the instructions from the manufacturer. Direct sequencing was performed on a 3500 xL Genetic Analyzer (Applied Biosystems, Foster City, CA, United States). All mutations were confirmed in a second, independent PCR experiment.
5
Amplified Sequence Verification of Histoplasma
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From the amplified real-time PCR products, 5% (n= 15) were purified using ExoSAP (Affymetrix, Santa Clara, CA) according to the manufacturer’s instructions. Cycle sequencing reactions (20 μL) were prepared with 2 µL of DNA template and 3.2 pmol of the same primers used for PCR, 2 µL of BigDye Terminator v3.1, 2 µL of sequencing buffer (Applied Biosystems, Inc., Waltham, MA) according to the manufacturer’s instructions. Extension products were purified using Centri-Sep plates (Princeton Separations, Inc., Freehold, NJ) and electrophoresed on a 3730 DNA analyzer (Applied Biosystems, Inc.). The obtained sequences were edited manually based on the chromatograms, and the mold and complementary sequences were identified using Geneious 11.0.2. Software. BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi ) was used to verify that the sequenced PCR products belonged to H. capsulatum.
6
Cloning and Sequencing of MHC Amplicons
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PCR products were purified using a GeneJet Gel Extraction Kit (Thermo Scientific™), and the purified amplicons were cloned into the pJET vector using the CloneJET PCR cloning kit, both according to the manufacturer’s guidelines (Thermo Scientific™). Next, the cloned amplicons were transformed in Escherichia coli XL1-blue cells by using the TransformAid Bacterial Transformation Kit (Thermo Scientific™). Per animal, 24 to 48 bacterial clones were picked, and plasmid DNA was isolated using a standard mini-preparation procedure. The purified plasmid DNA was sequenced on the ABI 3500 genetic analyser (Applied Biosystems, Foster City, USA). The sequencing reaction was performed by using 2 μM pJET primer, 1 μL BigDye terminator, and 2 μL of 5 × sequencing buffer in a total volume of 10 μL (Thermo Scientific™). The resulting sequences were analysed using the Sequence Navigator programme (Applied Biosystems, Foster City, USA). MHC sequences were revised manually by applying the Lasergene 12 SeqMan Pro Sequence Alignment Editor.
7
Identification of Quinolone Resistance in Klebsiella pneumoniae and Escherichia coli
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QRDR regions of gyrA gene of both clinical isolate CIP-resistant Klebsiella pneumoniae (Kp8) and CIP-resistant Escherichia coli (E17) were amplified by PCR from the chromosomal DNA of quinolone-resistant isolates. A 344-bp region covering the QRDR of gyrA was amplified with primers 5′-AAATCTGCCCGTGTCGTTGGT-3′and 5′- GCCATACCTACTGCGATACC-3′ [50 (link)]. PCR was performed as recommended, and PCR products were visualized by 1% agarose gel electrophoresis using ethidium bromide [51 (link)]. PCR products were purified with high pure PCR product purification kits (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany). Purified PCR products were sequenced using Applied Biosystems 3500 Genetic Analyzer (Hitachi, ThermoFisher, MA, USA) in Colors labs, Egypt. The primers used for sequencing were the same used for amplification. BigDye Terminator v3.1 Cycle Sequencing Kit and 5× Sequencing Buffer (ThermoFisher) were used.
8
Sequencing and analysis of TP53, BRCA1, and BRCA2
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For TP53, the different melting curve fragments were sequenced in an automatic sequencer XL 3500 Genetic Analyzer (Applied Biosystems). The reaction consisted of 1 to 2 uL of amplified DNA, 2 uL of Big Dye Terminator v3.1 Cycle, 2 uL of 5X Sequencing Buffer (Applied Biosystems), 1 uL of primer and sufficient water to complete 10 uL. The amplification parameters of the sequencing reaction were: 95°C for 1 minute followed by 25 cycles of 95°C for 10 seconds, 50°C for 5 seconds and 60°C for 4 minutes.
The complete coding sequence and exon-intron boundaries of BRCA1 and BRCA2 were analyzed in two TP53 p.R337H positive females. The sequences of the primers were those described by Leeneer et al. [23 (link)], and Keshavarzi et al. [24 (link)], respectively.
The complete coding sequence and exon-intron boundaries of BRCA1 and BRCA2 were analyzed in two TP53 p.R337H positive females. The sequences of the primers were those described by Leeneer et al. [23 (link)], and Keshavarzi et al. [24 (link)], respectively.
9
Sanger Sequencing of CEL VNTR Region
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After standard treatment of the CEL exon 8-11 PCR product with ExoSap-IT reagent (Thermo Fisher Scientific), Sanger sequencing of the VNTR region was performed with the primers EF (5´- CACACACTGGGAACCCT-3´) and VNTR-R (5´-TCCTGCAGCTTAGCCTTGGG-3´). The reaction mix (10 μL) consisted of 2.0 μL of the treated PCR product, 0.25 μL primer (20 μM), 2.0 μL betaine (5 M), 2.0 μL 5X Sequencing Buffer (Applied Biosystems), 2.75 μL ddH2O, and 1.0 μL BigDye Terminator v3.1 reagent (Applied Biosystems). Amplification consisted of an initial denaturation step at 96 °C for 10 minutes, followed by 25 cycles of denaturation at 96 °C for 10 seconds, annealing at 58 °C for 5 seconds and elongation at 60 °C for 4 minutes. The fluorescent-labeled sequencing products were purified using Sephadex G-50 Superfine (Sigma) and analyzed by capillary electrophoresis on a 3500xL Genetic Analyzer (Applied Biosystems). All sequences were evaluated manually with the help of the software SnapGene Version 4.1.9 (www.snapgene.com ). CEL VNTR lengths were determined using a published method (11 (link)), using only the unlabeled forward primer Celex11F (5´-ACCGACCAGGAGGCCACCC-3´) and the fluorescently (NED) labeled reverse primer Celex11R-NED (5´-CCTGGGGTCCCACTCTTGT-3´).
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