Total RNA was extracted using an ISOGEN kit (NIPPON GENE, Tokyo, Japan), and converted to cDNA using Superscript III and random primers (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The Dll fragment was PCR amplified from the cDNA using a set of primers designed from the Dll sequence retrieved from wFleaBase http://wfleabase.org/ (Additional file 2 : Table S1). Subsequently, the cDNA fragments were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Plasmids were sequenced using Sanger techniques that included the Big Dye terminator Ver. 3.1 (Life Technologies) on an ABI 3100 Avant or ABI 3130 Genetic Analyser DNA sequencer (Applied Biosystems Japan Ltd, Tokyo, Japan).
Abi 3100 avant
Manufactured by Thermo Fisher Scientific
Sourced in Japan, United States, Germany
The ABI 3100 Avant is a capillary electrophoresis genetic analyzer designed for DNA sequencing and fragment analysis applications. It features 16 capillaries and can process multiple samples simultaneously. The instrument utilizes laser-induced fluorescence detection technology to analyze DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Platinum taq polymerase, by Thermo Fisher Scientific (2 mentions)
Bigdye terminator ready reaction cycle sequencing kit v 3, by Thermo Fisher Scientific (2 mentions)
3500 genetic analyser, by Thermo Fisher Scientific (2 mentions)
Pcr purification kit, by Qiagen (2 mentions)
Salsa mlpa p256 flcn probemix, by MRC-Holland (2 mentions)
Pgem t easy vector, by Promega (1 mentions)
Random primer, by Thermo Fisher Scientific (1 mentions)
Isogen kit, by Nippon Gene (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Pcr4blunt topo, by Thermo Fisher Scientific (1 mentions)
Lb broth, by Merck Group (1 mentions)
Ampicillin, by Fujifilm (1 mentions)
Coffalyser, by Thermo Fisher Scientific (1 mentions)
Net software, by MRC-Holland (1 mentions)
Hotstartaq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Coffalyser net software, by MRC-Holland (1 mentions)
Genemapper software, by Thermo Fisher Scientific (1 mentions)
7 protocols using abi 3100 avant
1
Cloning and Sequencing of Dll Gene
2
Sequencing of ATP6AP1 Gene in Patients
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Genomic DNA was extracted from fibroblast pellets or white blood cells from 10 patients and available family members. Primers (Supplementary Table 4 ) were designed to amplify the 10 exons of ATP6AP1 (GenBank accession number NM_001183.4), including at least 50 bp of the flanking intronic regions. Standard PCR reactions were based on 1 μl DNA and 0.2 μl Platinum Taq polymerase (Invitrogen) in a total volume of 25 μl. Standard reaction conditions were 10 min at 95 °C, then 35 cycles of 30 s at 95 °C, 30 s at 60 °C and 1 min at 72 °C. The reaction was completed with a final elongation of 7 min at 72 °C. For the sequencing of the resulting PCR product, the BigDye Terminator Ready reaction cycle sequencing kit v.3.1 (Applied Biosystems) was used. Analysis of the results was performed on an ABI3100 Avant (Applied Biosystems).
3
Validation of Genetic Variants by Sanger Sequencing
4
Plasmid Purification and Sequencing from S. aureus
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Purification of plasmids from S. aureus strains positive for ses and set was carried out as described
previously [16 (link), 18 (link)]. The TOPO shotgun cloning kit (Invitrogen, Carlsbad, CA, USA),
pCR4®Blunt-TOPO® (Invitrogen) and One shot® TOPO10 electrocompTME. coli (Invitrogen) were used
for preparation of the shotgun library. White colonies were cultured in LB broth (Sigma Aldrich, St. Louis, MO, USA) containing 100 µg/ml ampicillin (Wako Pure
Chemical Industries, Osaka, Japan) and cultured at 37°C under shaking conditions overnight. The cultured cells were subjected to plasmid extraction using the
QIAprep8 (QIAGEN, Hilden, Germany) system. Nucleotide sequences of the shotgun library were obtained using an automatic DNA sequencer ABI3100Avant (Applied
Biosystems, Foster City, CA, USA) and assembled using AGCT software, ver. 4.0 (Genetyx, Tokyo, Japan). Gaps of contigs were closed by primer walking with
primers designed at contig ends.
previously [16 (link), 18 (link)]. The TOPO shotgun cloning kit (Invitrogen, Carlsbad, CA, USA),
pCR4®Blunt-TOPO® (Invitrogen) and One shot® TOPO10 electrocompTME. coli (Invitrogen) were used
for preparation of the shotgun library. White colonies were cultured in LB broth (Sigma Aldrich, St. Louis, MO, USA) containing 100 µg/ml ampicillin (Wako Pure
Chemical Industries, Osaka, Japan) and cultured at 37°C under shaking conditions overnight. The cultured cells were subjected to plasmid extraction using the
QIAprep8 (QIAGEN, Hilden, Germany) system. Nucleotide sequences of the shotgun library were obtained using an automatic DNA sequencer ABI3100Avant (Applied
Biosystems, Foster City, CA, USA) and assembled using AGCT software, ver. 4.0 (Genetyx, Tokyo, Japan). Gaps of contigs were closed by primer walking with
primers designed at contig ends.
5
Genetic Screening of FLCN Gene
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The complete FLCN gene coding region including adjacent intronic sequences was screened for mutations by PCR and subsequent Sanger sequencing following standard protocols (for primer details and PCR protocols see S1 Table ). For each PCR 50–100 ng DNA were amplified using the HotStarTaq DNA Polymerase and InvitrogenTMTaq DNA Polymerase recombinant (Qiagen, Hilden, Germany; Thermo Fisher Scientific, Dreieich, Germany). Purification of the amplification products was performed with the Qiagen PCR purification kit (Qiagen, Hilden, Germany), and PCR products were sequenced using the 3500 Genetic Analyser (Thermo Fisher Scientific, Dreieich, Germany). MLPA (multiplex ligation-dependent probe amplification) was performed using the SALSA MLPA P256 FLCN probemix (MRC Holland, Amsterdam, The Netherlands) according to the manufacturer’s protocol on the ABI 3100 Avant (Applied Biosystems, Darmstadt, Germany) and analyzed by Coffalyser.Net software (MRC Holland, Amsterdam, The Netherlands). Statistical analyses were performed using the two-tailed Mann-Whitney U Test and the Chi-square Test.
6
FLCN Gene Mutation Screening Protocol
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The coding region of the FLCN gene including adjacent intronic sequences was amplified by PCR and analyzed by Sanger sequencing following standard protocols.4 In summary, PCR amplification was performed with 50-100 ng DNA using HotStarTaq DNA Polymerase or Invitrogen™Taq DNA Polymerase recombinant (Qiagen, Hilden, Germany; Thermo Fisher Scientific, Dreieich, Germany). PCR products were prepared for sequencing with the Qiagen PCR purification kit (Qiagen, Hilden, Germany), and Sanger sequencing was performed using the 3500 Genetic Analyser (Thermo Fisher Scientific, Dreieich, Germany). For MLPA the SALSA MLPA P256 FLCN probemix (MRC Holland, Amsterdam, The Netherlands) was used on the ABI 3100 Avant (Applied Biosystems, Darmstadt, Germany) and analyzed by Coffalyser Net software (MRC Holland, Amsterdam, The Netherlands).22
7
Genetic Diversity of Indian Goat Breeds
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The present research work was conducted at National Bureau of Animal Genetics Resources, Karnal-132001 during 2016-17. The data on 25 STR markers generated for 22 goat breeds had already been published by Dixit et al. (2012) . Briefly, 1034 random blood samples (36-48 samples from each breed) were collected from different parts of the country and DNA was isolated using standard protocol. The denatured samples were run on automated DNA sequencer of Applied Biosystems (ABI 3100 Avant). The electropherograms drawn through Gene Scan were used to extract DNA fragment sizing details using Gene Mapper software (version 3.0) (Applied Biosystems, U.S.A.). The breed-wise population data of 24 registered goat breeds was also collected from estimated livestock population breed-wise survey-2013, Department of Animal Husbandry, Government of India. The other relevant information required was based on review of literature viz., breed wise monographs published by National Bureau of Animal genetic Resources, Karnal; Indian J. of animal Sci. vol. 77-83; 19 th livestock census, Department of Animal Husbandry, Government of India; Network and NATP project reports of National Bureau of Animal Genetic Resources, Karnal; Scientist opinion and farmers talk and personal experience of the authors with goat farmers.
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