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Genescan 1200 liz

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The Genescan 1200 LIZ is a fragment analysis system designed for accurate size determination of DNA fragments. It utilizes capillary electrophoresis technology to separate and detect fluorescently labeled DNA samples. The instrument can be used to analyze a variety of DNA fragment types, including microsatellites, short tandem repeats, and amplified fragment length polymorphisms.

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Lab products found in correlation

3130 genetic analyzer, by Thermo Fisher Scientific (2 mentions) Genemapper software, by Thermo Fisher Scientific (2 mentions) Megabace tmet, by GE Healthcare (2 mentions) Hi di formamide, by Thermo Fisher Scientific (1 mentions) Abi 3730 sequencer, by Thermo Fisher Scientific (1 mentions) Genemarker software, by SoftGenetics (1 mentions) Amplitaq gold 360 master mix, by Thermo Fisher Scientific (1 mentions) Abi 3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions) Abi 3730xl capillary, by Thermo Fisher Scientific (1 mentions) Genescan 500 liz, by Thermo Fisher Scientific (1 mentions) Abi 3500, by Thermo Fisher Scientific (1 mentions) Genemapper, by Thermo Fisher Scientific (1 mentions) Abi 3130 l genetic analyzer, by Thermo Fisher Scientific (1 mentions) Haeiii, by Takara Bio (1 mentions)

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GeneScan 1200 LIZ size standard (17 citations) GeneScan™ 1200 LIZ™ dye Size Standard (3 citations)

9 protocols using genescan 1200 liz

1

Standardized Anthrax Genotyping via MLVA

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MLVA is a standard tool for B. anthracis genotyping [10 (link)]. Since most of the long and highly-repetitive VNTRs cannot not be determined by whole genome sequencing, lengths of amplified repetitive regions were analyzed by DNA-fragment-length analysis supported in part by Sanger sequencing, allowing for the calculation of repeat numbers or definite base-sequence, respectively. Fragment lengths of fluorescently labeled amplicons covering the whole VNTR locus were measured by capillary gel electrophoresis. In order to amplify the 31 different VNTR loci five conventional multiplex PCRs were carried out as described previously [40 (link),41 (link),42 (link)] with minor changes in primer concentrations.
PCR products were diluted 1:10 in water. A volume of 2 μl of each dilution and 0.5 μl of size standard Genescan 1200 LIZ (Applied Biosystems) were added to 17.5 μl Hi-Di Formamide (Applied Biosystems) and incubated at 95°C for 5 min to denature DNA. Fragment lengths were analyzed on the 3130 Genetic Analyzer (Applied Biosystems) and the associated GeneMapper software.
Braun P., Grass G., Aceti A., Serrecchia L., Affuso A., Marino L., Grimaldi S., Pagano S., Hanczaruk M., Georgi E., Northoff B., Schöler A., Schloter M., Antwerpen M, & Fasanella A. (2015). Microevolution of Anthrax from a Young Ancestor (M.A.Y.A.) Suggests a Soil-Borne Life Cycle of Bacillus anthracis. PLoS ONE, 10(8), e0135346.
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2

Capillary Sequencer-based PCR Ribotyping Protocol

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Capillary sequencer-based PCR ribotyping was performed as described by Indra et al.44 (link). Generally, primers 16S-F (5′-GTGCGGCTGGATCACCTCCT-3′) and 23S-R (5′-CCCTGCACCCTTAATAACTTGACC-3′) were used for amplification, with 16S primer labeled at the 5′ end with carboxyfluorescein (FAM). The PCR product was diluted 1:10 in water before loading. Fragment separation was performed on an ABI 3730 sequencer (Applied Biosystems, Carlsbad, USA) with a 50 cm POP 7 gel. Sample injection was at 1.6 kV over 15 seconds, with a total running time of 6200 seconds. A GeneScan™ 1200 LIZ (Applied Biosystems) size standard was used as internal marker. The results were analyzed by GeneMarker software (Version2.2.0, SoftGenetics, State College, PA, USA), and queried against WEBRIBO database (https://webribo.ages.at/) for ribotypes. A previously well-characterized PCR ribotype 027 isolate, strain CA2, was used as internal control for PCR ribotyping10 (link).
Cheng J.W., Xiao M., Kudinha T., Xu Z.P., Hou X., Sun L.Y., Zhang L., Fan X., Kong F, & Xu Y.C. (2016). The First Two Clostridium difficile Ribotype 027/ST1 Isolates Identified in Beijing, China–an Emerging Problem or a Neglected Threat?. Scientific Reports, 6, 18834.
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3

Mycobacterial MIRU-VNTR Genotyping Protocol

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The primer sets proposed by Dauchy et al [10] (link) and Iakhiaeva et al [11] (link) were used, except MIRU 3, which was not amplified in this study. PCR mixtures were prepared using 10 μL of AmpliTaq Gold 360 master mix (Applied Biosystems), 10 ng of template DNA, and 4–7.5 μM of primers (Table 2). The PCR conditions were as follows: 5 minutes at 94°C, followed by 30 cycles at 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute, with a final extension step at 72°C for 7 minutes.
The size of amplified products was analyzed on ABI 3730XL DNA analyzer (Applied Biosystems). GeneScan 1200 LIZ (Applied Biosystems) was used for fragment analysis.
Jeon S., Lim N., Kwon S., Shim T., Park M., Kim B.J, & Kim S. (2014). Molecular Typing of Mycobacterium intracellulare Using Pulsed-Field Gel Electrophoresis, Variable-Number Tandem-Repeat Analysis, Mycobacteria Interspersed Repetitive-Unit-Variable-Number Tandem Repeat Typing, and Multilocus Sequence Typing: Molecular Characterization and Comparison of Each Typing Methods. Osong Public Health and Research Perspectives, 5(3), 119-130.
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4

Profiling SNR lengths via fragment analysis

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Because of intrinsic technical-limitations of Ion Torrent technology the results of whole genome sequencing cannot be used for analysis of the highly mutable long homopolymeric regions. Therefore, fragment length analysis was employed to determine SNR lengths [13 (link)] (Table 2). Each of the four SNR-loci (HM1, 2, 6 or 13) were analyzed in single- or multi-plex PCR reactions. PCR products were diluted, denatured and run on the 3130 Genetic Analyzer (Applied Biosystems) with size standard Genescan 1200 LIZ (Applied Biosystems) using the associated GeneMapper software.
Braun P., Grass G., Aceti A., Serrecchia L., Affuso A., Marino L., Grimaldi S., Pagano S., Hanczaruk M., Georgi E., Northoff B., Schöler A., Schloter M., Antwerpen M, & Fasanella A. (2015). Microevolution of Anthrax from a Young Ancestor (M.A.Y.A.) Suggests a Soil-Borne Life Cycle of Bacillus anthracis. PLoS ONE, 10(8), e0135346.
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5

MLVA Genotyping of B. anthracis

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The MLVA was performed essentially as described in [16 (link)]. Briefly, amplification of the fragments of 31 marker-loci was performed in 7 multiplex-PCRs (the origins of the best matches to the Finnish B. anthracis isolates are listed in Additional file 2). The fragment-mixtures were analyzed on a Genetic Analyzer (ABI 3130, Applied Biosystems, Germany) using either MegaBACE TMET (GE Healthcare, Germany), Genescan 1200 LIZ (Applied Biosystems, USA) or MapMarkerH 1000 (BioVentures, USA) as size standards. The data were analyzed with GeneMapper TM software (Applied Biosystems, USA). The raw data of fragment lengths were normalized by codes, reflecting the actual copy numbers of the repeat sequences where possible.
Lienemann T., Beyer W., Pelkola K., Rossow H., Rehn A., Antwerpen M, & Grass G. (2018). Genotyping and phylogenetic placement of Bacillus anthracis isolates from Finland, a country with rare anthrax cases. BMC Microbiology, 18, 102.
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6

Screening Wheat Lines for Stem Rust Resistance

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DNA was extracted from single wheat seedlings using the CTAB extraction method (Doyle and Doyle, 1990 ). All 256 lines were screened with published markers for 11 stem rust resistance genes using the published PCR protocols (Supplementary Table 2). Fluorescently labeled primers allowed sequence-based fragment detection on an ABI3730xl capillary instrument (Applied Biosystems, Foster City, CA, USA) applied at the Central Analytical Facility of Stellenbosch University, South Africa. GeneScan™ 500 LIZ® or GeneScan™ 1200 LIZ® (Applied Biosystems) was used as an internal size standard. Data were analyzed using GeneMapper v4.0 (Thermofischer, formally Applied Biosystems). A stem rust resistance gene was called as present if the marker-allele at all markers associated with the Sr gene were the same as the control line. Controls for known stem rust resistance genes and gene complexes included: Cranbrook (Sr2), Sr22B (Sr22), Palmiet (Sr2, Sr24), Avocet S (Sr26), Federation*4/Kavkaz (Sr31), RL5405 (Sr33), Mq12/5*G2191_Rsr35 (Sr35), SrTt1Sr36 (Sr36), RL6082 (Sr39/Lr35), Kariega (Lr34/Yr18/Sr57) and Pavon 76 (Lr46/Yr29/Sr58).
Prins R., Dreisigacker S., Pretorius Z., van Schalkwyk H., Wessels E., Smit C., Bender C., Singh D, & Boyd L.A. (2016). Stem Rust Resistance in a Geographically Diverse Collection of Spring Wheat Lines Collected from Across Africa. Frontiers in Plant Science, 7, 973.
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7

MLVA Genotyping Protocol for Bacterial Strain Identification

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MLVA was performed essentially as described in [32 (link)]. Amplification of fragments of 31 marker loci was performed in 7 multiplex PCR. Fragment mixtures were analyzed on a genetic analyzer (ABI 3500, Applied Biosystems, USA) using Genescan 1200 LIZ (Applied Biosystems, USA) as size standards. The data were analysed using the software GeneMapper TM (Applied Biosystems, USA). The MLVA31 cluster analysis was performed using BioNumerics version 7.6 software package (Applied Maths, Belgium). The minimum spanning tree was built based on the number of tandem repeats using the categorical distance coefficient.
Pisarenko S.V., Eremenko E.I., Ryazanova A.G., Kovalev D.A., Buravtseva N.P., Aksenova L.Y., Evchenko A.Y., Semenova O.V., Bobrisheva O.V., Kuznetsova I.V., Golovinskaya T.M., Tchmerenko D.K., Kulichenko A.N, & Morozov V.Y. (2019). Genotyping and phylogenetic location of one clinical isolate of Bacillus anthracis isolated from a human in Russia. BMC Microbiology, 19, 165.
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8

T-RFLP Analysis of Archaeal and Bacterial 16S rRNA

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Archaeal and bacterial 16S rRNA gene fragments for T-RFLP analysis were amplified by PCR using the primer pairs 5′ FAM-labeled Arch21F/Ar912r and 5′ FAM-labeled EUB338F/1492R, respectively. PCR was performed under the conditions described above. The PCR products were digested with HaeIII and HhaI (TaKaRa Bio Inc., Otsu, Japan) separately. The labeled fragments were analyzed by electrophoresis on an ABI 3130×l Genetic Analyzer (Applied Biosystems, CA, USA). GeneScan 1200 LIZ (Applied Biosystems) was used as a size standard.
Aoki M., Ehara M., Saito Y., Yoshioka H., Miyazaki M., Saito Y., Miyashita A., Kawakami S., Yamaguchi T., Ohashi A., Nunoura T., Takai K, & Imachi H. (2014). A Long-Term Cultivation of an Anaerobic Methane-Oxidizing Microbial Community from Deep-Sea Methane-Seep Sediment Using a Continuous-Flow Bioreactor. PLoS ONE, 9(8), e105356.
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9

MLVA Typing of Bacterial Samples

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MLVA was done as described previously [24 (link)]. Briefly, amplification of the fragments of 31 marker-loci was performed in seven multiplex-PCRs. The fragment mixtures were analyzed on a Genetic Analyzer (ABI 3130, Applied Biosystems, Darmstadt, Germany) using either MegaBACE TMET (GE Healthcare, Solingen, Germany), Genescan 1200 LIZ (Applied Biosystems Darmstadt, Germany) or MapMarkerH 1000 (BioVentures, Murfreesboro, TN, USA) as size standards. The data were analyzed with GeneMapper TM software (Applied Biosystems, Darmstadt, Germany). The raw data of fragment lengths were normalized by codes, reflecting the actual copy numbers of the repeat sequences where possible. MLVA data analysis was performed using cluster analysis of categorical coefficients. Herein, a similarity tree based on the unweighted pair group method with arithmetic mean (UPGMA) method was computed in BioNumerics 6.6 (Applied Maths, Sint-Martens-Latem, Belgium) and manually edited (using Powerpoint, Microsoft) for style.
Antwerpen M., Beyer W., Bassy O., Ortega-García M.V., Cabria-Ramos J.C., Grass G, & Wölfel R. (2019). Phylogenetic Placement of Isolates Within the Trans-Eurasian Clade A.Br.008/009 of Bacillus anthracis. Microorganisms, 7(12), 689.
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