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 of Taq FIREPol^® DNA polymerase (Solis Biodyne FIREPol^®, catalog number: 01-01-01000 ; any other suppliers should be also satisfactory) with the corresponding 10x PCR buffer (supplied with FIREPol^® DNA polymerase)
      
   2. 25 mM MgCl₂ (supplied with FIREPol^® 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 (1µM) (5´- CACGACGTTGTAAAACGAC-3´) (desalted) (Microsynth)
      
   5. Reverse specific primer (10 µM) (desalted) (Microsynth)
      
   6. Fluorescence (6-FAM, HEX, NED or Cy5, D2, D3) labeled primer with a complementary sequence to the M13 tail (10 µM) (Metabion, Planegg/Steinkirchen)
3. Restriction analysis
   
   1. Restriction endonucleases (New England Biolabs or Fermentas)
      
   2. 1x Buffer restriction endonucleases (New England Biolabs or Fermentas)
4. 1.5% agarose (Sigma-Aldrich, catalog number: A9539 )
   
5. Ethidium bromide (Roche Diagnostics, catalog number: HP46.2 )
   
6. DNA loading buffer 3x [6x; 30% (v/v) glycerol, 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol FF]
   
7. Analysis on ABI PRISM 3100 Genetic Analyzer Separation Gel (Applied Biosystems, catalog number: 4363929 )
   
   1. ROTISOLV^® HPLC gradient grade water (Roche Diagnostics, catalog number: A511.3 )
      
   2. HiDi^TMFormamide as Sample Loading Buffer (Applied Biosystems, catalog number: 4311320 )
      
   3. GeneScan^TMROX size standard (Applied Biosystems, catalog number: 401734 )
      
   4. 96 well half skirted PCR plates (Kisker, catalog number: G060/H/1E/OA-SS )
8. Analysis on Beckman Coulter CEQ^TM 8000 Genetic Analysis System
   
   1. GenomeLab^TM Separation Gel LPA I (Beckman Coulter, catalog number: 608010 )
      
   2. 96 well half skirted, segmented PCR plates (Kisker, catalog number: G060/H/1E-SG )
      
   3. Separation Buffer plates, non-sterile (Beckman Coulter, catalog number: 609844 )
      
   4. GenomeLab^TM DNA Size Standard 600 (Beckman Coulter, catalog number: 608095 )
      
   5. GenomeLab^TM Separation Buffer (Beckman Coulter, catalog number: 608012 )
      
   6. Formamide as Sample Loading Buffer (Sigma-Aldrich, catalog number: F9037 ) or original Sample Loading Buffer (SLS) (Beckman Coulter, catalog number: 608082)
9. Gel running buffer (see Recipes)
   

Equipment

1. Thermal Cycler (Applied Biosystems)
   
2. Horizontal Electrophoresis system (Bio-Rad Laboratories, Sub-Cell^® Model 96 Cell)
   
3. ABI PRISM 3100 Genetic Analyzer or Beckman Coulter CEQ^TM 8000 Genetic Analysis System
   
4. Heating block, bath or oven for restriction analysis
   
5. PCR 96-well plate or tubes
   

Software

1. GeneMapper^® 4.0 Software (Applied Biosystems) or GenomeLab^TM GeXP Software (Beckman Coulter)

Procedure