High-throughput Method for Determination of Seed Paternity by Microsatellite Markers
利用微卫星标记法高通量鉴定种子亲权
发布: 2013年04月20日第3卷第8期 DOI: 10.21769/BioProtoc.452 浏览次数: 13102
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参见作者原研究论文
The authors used this protocol in:
Aug 2012
Abstract
In this protocol, determination of seed paternity by microsatellite markers in Nicotiana attenuata is described. However, this does not include a protocol for the novel marker selection/identification, but rather exploits the markers generated for a closely related species N. tabacum (Bindler et al., 2007). This is a high-throughput protocol optimized and streamlined for one skilled person to process 384 (96 x 4) seeds in 5 days, from DNA isolation (from seedlings) to paternity assessment by microsatellite genotype data.
Keywords: Microsatellite genotyping (微卫星基因分型)
Multiplex PCR (多重PCR)
High-throughput seed paternity test (高通量种子亲子鉴定试验)
Nicotiana attenuata (野生烟草)
Materials and Reagents
- Agencourt Chloropure Kit (this kit is discontinued by Beckman Coulter but can be ordered) or Quiagen MagAttract 96 DNA plant core kit (QIAGEN, catalog number: 67163 )
- Qiagen Type-it Multiplex PCR kit (QIAGEN, catalog number: 206243 )
- QIAquick PCR purification kit (QIAGEN, catalog number: 28106 )
- Liquid nitrogen
- 100% Isopropanol, ultrapure
- Freshly prepared 70% ethanol made with nuclease free water
Note: 70% ethanol is hygroscopic, always make fresh for optimal result. - Agarose
- Ethydium bromide solution
- Nuclease free water
- Pure molecular biology grade ethanol (96– 100%)
Note: Calculate the volume of ethanol needed before you start. A new kit of 24 plates needs about 5 L of ethanol. - GENSCAN 500 ROX (Applied Biosystems, catalog number: 401734 )
- 1x TAE (Tris-Acetate-EDTA) buffer (see Recipes)
Equipment
- 2.2 ml Ritter Deep-well plates (ABGene, catalog number: AB-6661 )
- Adhesive plate film (ABGene, catalog number: AB-0558 , AB-626 , AB-662 )
- 4 mm steel balls (SPEX SamplePrep, catalog number: 2150 )
- Pipette tips
- Processsing Plate: Deep-well titer plate with a 96-well format (SPEX SamplePrep, catalog number: 2210 )
- Destination Plate: 300 μl round bottom microtiter plate (96 well, 300 μl well capacity, round bottom) (Corning, Costar®, catalog number: 07-200-105 )
- Desktop centrifuge for 96 well plates
- Reagent reservoir
- Genogrinder (SPEX SamplePrep, catalog number: 2010 )
- Cooling block (SPEX SamplePrep, catalog number: 2665 )
- Steel ball dispenser (SPEX SamplePrep, catalog number: 2100 )
- Multi-channel pipettes (0.1-10, 10-100, 30-300 μl)
- Agencourt Supermagnet magnetic Plate (Agencourt SPRIPlate 96R-Ring Magnet Plate, catalog number: A29164 )
- BioRad Gel casting tray, running tray, power pack etc (Bio-Rad Laboratories)
- Nanodrop spectrophotometer (Nanodrop)
- Pipette (0.2-2 μl), pipette tips, soft tissue paper
- ABI 3100 Genetic Analyzer (Applied Biosysyems)
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Bhattacharya, S. and Baldwin, I. T. (2013). High-throughput Method for Determination of Seed Paternity by Microsatellite Markers. Bio-protocol 3(8): e452. DOI: 10.21769/BioProtoc.452.
Download Citation in RIS Format
分类
分子生物学 > DNA > 基因分型
植物科学 > 植物分子生物学 > DNA
植物科学 > 植物生理学 > 组织分析
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