Genomic DNA Extraction and Genotyping of Dictyochloropsis Green Algae Strains
Dictyochloropsis绿藻菌株的基因组DNA提取和基因分型
发布: 2015年08月05日第5卷第15期 DOI: 10.21769/BioProtoc.1545 浏览次数: 12604
评审: Arsalan DaudiAntoine DanonClaudia Catalanotti
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参见作者原研究论文
The authors used this protocol in:
Apr 2014
Abstract
Dictyochloropsis is an ecologically important genus of free-living and symbiotic green algae. Representatives of this genus are horizontally transmitted among several fungi of the family Lobariaceae, thus forming photobiont-mediated guilds. This protocol is suitable for extracting DNA from algal cultures and lichen samples and for genotyping seven unlinked Dictyochloropsis reticulata microsatellite markers in a single PCR multiplex.
Figure 1. Schematic representation of the analysis pipeline
Materials and Reagents
- Qiagen Type-it Microsatellite PCR kit (QIAGEN, catalog number: 206243 )
- 1x TE buffer (10 mM Tris, bring to pH 8.0 with HCl, 1 mM EDTA)
- GelRed (Biotium, catalog number: 41003 )
- 1 kb DNA ladder (GeneRuler 1 kb DNA Ladder) (Thermo Fisher Scientific, catalog number: SM0313 )
- Nuclease-free water
- Pure molecular biology grade ethanol (96–100%)
- 2 ml tubes
- DNeasy 96 Plant Kit (QIAGEN, catalog number: 69181 )
- Hi-Di™ formamide (Life Technologies, catalog number: 4311320 )
- GeneScan 500 LIZ size standard (Life Technologies, catalog number: 4322682 )
- Ice
- Glucose (BD Biosciences, catalog number: 215510 )
- Proteose pepton (BD Biosciences, catalog number: 212230 )
- Agar (BD Biosciences, catalog number: 214883 )
- 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) (Life Technologies, Gibco®, catalog number: 11330032 )
- Laboratory wipes (e.g., KimWipes, Kimberly-Clark, catalog number: TW31KWPBX )
- 4 mm steel balls (e.g., Spex Certiprep, catalog number: 12145950 )
- Pipette tips
- Pipette (0.1-2 μl), pipette tips
- Processing plate: 96-well plate (e.g., MicroAmp Optical 96-Well Reaction Plate) and 96-well plate (Septa)
- 0.22 µm sterile polyethersulfon syringe filter (Merck Millipore, catalog number: P/N SLMP025SS )
- Algal medium (see Recipes)
- Algal culture medium preparation for growing Dictyochloropsis and other trebouxiophycean algal strains (see Recipes)
- Vitamin solution (see Recipes)
Equipment
- Multi-channel pipettes (0.1-10, 10-100, 100-1000 μl), with extended tips
- Microcentrifuge with rotor for 2 ml tubes
- Centrifuge for 96-well plates
- BioRad Gel casting tray, running tray, power pack etc. (Bio-Rad Laboratories, catalog number: 164-0305 )
- Incubator (65 °C)
- Freezer or cold room at -20 °C
- PCR Thermal cycler
- ABI3130/ 3130xl/3730/3730xl DNA Analyzer (Applied Biosystems)
- Ball Mill MM 400 (Retsch®, catalog number: 20.745.0001 )
- Lyophilizer (e.g., FreeZone 4.5 Liter Benchtop Freeze Dry System, Labconco, catalog number: 7750021 )
Software
- GeneMapper® (Software v4.1, Applied Biosystems, catalog number: 4366925)
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Grande, F. D., Cornejo, C., Scheidegger, C. and Beck, A. (2015). Genomic DNA Extraction and Genotyping of Dictyochloropsis Green Algae Strains. Bio-protocol 5(15): e1545. DOI: 10.21769/BioProtoc.1545.
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分类
植物科学 > 藻类学 > DNA
植物科学 > 植物分子生物学 > DNA
分子生物学 > DNA > 基因分型
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