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Wheat Genetics Resource Center

Kansas State University
Department of Plant Pathology

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1712 Claflin Road
Manhattan, KS 66506-5502
785-532-6176 (TEL)
785-532-5692 (FAX)
wgrc@k-state.edu

Wheat Genetics Resource Center
Kansas Wheat Innovation Center

1990 Kimball Ave
Manhattan, KS 66502
785-320-4383 (TEL)

 

KSUD14-STS ASSAY FOR TAGGING LR21

New D14 primers.

Lr21L:

CGC TTT TAC CGA GAT TGG TC

Lr21R:

TCT GGT ATC TCA CGA AGC CTT

Lr21: 669 bp

lr21-Fielder: 757 bp

lr21-Wichita: 774 bp

Recipe (in 25 µl reaction). 
Buffer (10X)2.5 µl
2.5 mM dNTP2.5 µl
25 mM MgCl2.5 µl
DNA polymerase (Applied Biosystems AmpliTaq Gold; part number N8080243)0.15 µl
Lr21L (1 pmol/ul)1.0 µl
Lr21R (1 pmol/ul)1.0 µl
DNA50.0 ng
ddH2Oto 25.0 ml
PCR program.
  1. 95°C for 8 minutes
  2. 95°C for 30 seconds
  3. 54°C for 1 minute
  4. 72°C for 1 minute
  5. repeat steps 2-4 34 times
  6. 72°C for 7 minutes
  7. 4°C to end
Gel electrophoresis.

Separate the PCR products on a 2% agarose gel at 50V for 1 hour on a medium size gel. See figure at right for diagnostic fragment for Lr21.

Gel electrophoresis of PCR products for Lr21 fragments.