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Quality traits. Waxy mutants

Codominant CAPS markers for the null allele Wx-A1b. 

contributed by Marcelo Helguera (mhelguera@correo.inta.gov.ar)

This codominant marker differentiates the normal Wx-A1a allele from the null Wx-A1b allele present in lines Komugi Nourin, Fugimi Komugi and the triple mutant THF7. After the PCR reaction a slight difference in product sizes can be observed, but the treatment with the restriction enzyme improves the visualization in an agarose gel. 

PCR protocol

Primers: 

     Wx-A1b-F-MH	5'-CCC CAA AGC AAA GCA GGA AAC-3'
     Wx-A1b-R-MH 	5'-CGG CGT CGG GTC CAT AGA TC-3'
PCR reaction:
  • MgCl2: 1.5 mM
  • dNTP: 200 µM each
  • DNA: 120 ng/reaction
  • Taq Polymerase (Promega): 1U/reaction (0.2µl/reaction)

final volume: 25 µl

PCR program:
  • Denaturing step: 94ºC, 3 min
  • Amplification step (40 cycles):
    • 94ºC, 45 sec
    • 55ºC, 30 sec
    • 72ºC, 60 sec
  • Extension step: 72ºC, 10 min
  • Hold at 4ºC
Digestion with Hind III (Promega):

Add 5U of the enzyme to 10 µl of the PCR reaction and incubate at 37°C for 1 h. 
  1. Kern
  2. Komugi Nourin
  3. THF7
  4. Fugimi Komugi
M: 100bp ladder, the 500bp fragment is more intense.

Conditions presented here should be consider only as a starting point of the PCR optimization for individual laboratories.
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