Materials and Reagents

1. DNA (25 ng/µl) e.g. from two-weeks old barley seedlings; or according to PCR practice of each particular organism
   
2. PCR reagents
   
   1. 0.5 U Taq DNA polymerase (Solis Biodyne FIREPol^®, catalog number: 01-01-01000 ) or HotStart Taq polymerase (Solis Biodyne HOT FIREPol^®, catalog number: 01-02-01000 ; any other suppliers should be also satisfactory) with the corresponding 10x PCR buffer (supplied with Taq DNA polymerase)
      
   2. 25 mM MgCl₂ (supplied with Taq DNA polymerase)
      
   3. dNTPs (10 µM each) (Thermo Fisher Scientific, catalog number: R0182 )
      
   4. Forward specific primer tailed at the 5´end with a universal M13 tail (5´- CACGACGTTGTAAAACGAC-3´) (desalted) (1 µM)
      
   5. Reverse specific primer (desalted) (10 µM)
      
   6. Biotinylated universal primer with a complementary sequence to the M13 tail (10 µM) (Metabion, Planegg/Steinkirchen)
3. Agarose (Sigma-Aldrich, catalog number: A9539 )
   
4. Ethidium bromide (Roche Diagnostics, catalog number: HP46.2 )
   
5. Pyrosequencing reagents
   
   1. Sequencing primer (desalted) (10 µM)
      
   2. Streptavidin Sepharose HP (GE Healthcare, catalog number: 17-5113-01 )
      
   3. Annealing buffer (QIAGEN, catalog number: 979009 ) (see Recipes)
      
   4. Binding buffer (QIAGEN, catalog number: 979006 ) (see Recipes)
      
   5. Denaturation solution (QIAGEN, catalog number: 979007 ) (see Recipes)
      
   6. Washing buffer (QIAGEN, catalog number: 979008 ) (see Recipes)
      
   7. High-purity water (Milli-Q)
      
   8. 70% ethanol
      
   9. PyroMark Gold Q96 Reagents (enzyme mixture, substrate mixture and nucleotides) (QIAGEN, catalog number: 972807 )
6. Gel running buffer (see Recipes)
   
7. 6x DNA loading buffer (see Recipes)
   

Equipment

1. Thermal Cycler (Applied Biosystems)
   
2. Horizontal Electrophoresis system (Bio-Rad Laboratories)
   
3. PyroMark Q96 instrument (QIAGEN, catalog number: 9001525 )
   
4. PyroMark Q96 plate (QIAGEN, catalog number: 979002 )
   
5. PyroMark Q96 Vacuum Workstation (220 V) (QIAGEN, catalog number: 9001529 )
   
6. Vacuum pump (KNF Lab, Typ N816.1.2 KN.18)
   
7. Orbital shaker for microtiter plates
   
8. Heating block (80 °C)
   
9. PyroMark Q96 Cartridge (QIAGEN, catalog number: 979004 )
   
10. PCR 96-well plate (Greiner Bio-One, catalog number: 652250 )
    

Software

1. PyroMark Assay Design Software V.1.0.6. (QIAGEN)

Procedure