High-throughput Method for Determination of Seed Paternity by Microsatellite Markers

Materials and Reagents

1. 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 )
   
2. Qiagen Type-it Multiplex PCR kit (QIAGEN, catalog number: 206243 )
   
3. QIAquick PCR purification kit (QIAGEN, catalog number: 28106 )
   
4. Liquid nitrogen
   
5. 100% Isopropanol, ultrapure
   
6. Freshly prepared 70% ethanol made with nuclease free water
   Note: 70% ethanol is hygroscopic, always make fresh for optimal result.
   
7. Agarose
   
8. Ethydium bromide solution
   
9. Nuclease free water
   
10. 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.
    
11. GENSCAN 500 ROX (Applied Biosystems, catalog number: 401734 )
12. 1x TAE (Tris-Acetate-EDTA) buffer (see Recipes)
    

Equipment

1. 2.2 ml Ritter Deep-well plates (ABGene, catalog number: AB-6661 )
   
2. Adhesive plate film (ABGene, catalog number: AB-0558 , AB-626 , AB-662 )
   
3. 4 mm steel balls (SPEX SamplePrep, catalog number: 2150 )
   
4. Pipette tips
   
5. Processsing Plate: Deep-well titer plate with a 96-well format (SPEX SamplePrep, catalog number: 2210 )
   
6. Destination Plate: 300 μl round bottom microtiter plate (96 well, 300 μl well capacity, round bottom) (Corning, Costar^®, catalog number: 07-200-105 )
   
7. Desktop centrifuge for 96 well plates
   
8. Reagent reservoir
   
9. Genogrinder (SPEX SamplePrep, catalog number: 2010 )
   
10. Cooling block (SPEX SamplePrep, catalog number: 2665 )
    
11. Steel ball dispenser (SPEX SamplePrep, catalog number: 2100 )
    
12. Multi-channel pipettes (0.1-10, 10-100, 30-300 μl)
    
13. Agencourt Supermagnet magnetic Plate (Agencourt SPRIPlate 96R-Ring Magnet Plate, catalog number: A29164 )
    
14. BioRad Gel casting tray, running tray, power pack etc (Bio-Rad Laboratories)
    
15. Nanodrop spectrophotometer (Nanodrop)
    
16. Pipette (0.2-2 μl), pipette tips, soft tissue paper
    
17. ABI 3100 Genetic Analyzer (Applied Biosysyems)
    

Procedure