MS-AFLP protocol (96 well plate)

© 2008 Christoph Grunau, Michael Blouin, Please cite: Blouin et al. "No evidence for large differences in genomic methylation between wild and hatchery steelhead (Oncorhynchus mykiss)" Can. J. Fish. Aquat. Sci. 67(2): 217?224 (2010) [REF]

This protocol was developed as part of a Master project of Virginie Thuiller. It is based on an AFLP protocol published by Hazen, S.P., Leroy, P., Ward, R. 2002. AFLP in Triticum aestivum L.: patterns of genetic diversity and genome distribution. Euphytica 125 (1): 89-102, 2002.

  • prepare high-quality DNA (e.g. by Proteinase K + phenole-chloroform extraction)
  • 1st day:
    • dilute DNA to 20 ng/µl (at least 22 µl per enzyme mix), mix well
    • prepare 22 DNA samples
    • NOTE: master mix are calculated with +10%
    • prepare digestion master mix HpaII-EcoRI:
    • 50x0.1 µl (1 U)HpaII5.5 µl
      50x0.12 µl (1 U)EcoRI6.6 µl
      50x4 µlbuffer NEB1 10x220 µl
      50x25.78 µlsterile destilled water1418 µl
    • prepare digestion master mix MspI-EcoRI:
    • 50x0.1 µl (2 U)MspI5.5 µl
      50x0.12 µl (1 U)EcoRI6.6 µl
      50x4 µlbuffer NEB2 10x220 µl
      50x25.78 µlsterile destilled water1418 µl
    • prepare digestion master mix AciI-EcoRI:
    • 50x0.1 µl (1 U)AciI5.5 µl
      50x0.12 µl (1 U)EcoRI6.6 µl
      50x4 µlbuffer NEB3 10x220 µl
      50x25.78 µlsterile destilled water1418 µl
    • mix well
    • distribute 30 µl of each master mix into 48 wells of a 96 well plate
    • add 10 µl of each DNA sample to the enzyme mix, do 2 restriction reactions for each DNA sample, mix by pipetting
    • as positive control add 10 µl of a DNA sample with a known profile into 2 wells
    • as negative control add 10 µl of water into 2 wells (total number of samples is 24 now)
    • seal with aluminum sheet, centrifuge shortly
    • incubate at 37°C for 2 to 3 hours
    • prepare AciI/HpaII/MspI linker/adaptators for 200 reactions:
    • AciI/HpaII Linker 1GACGATGAGTCTAGAA100 µM100 µl
      AciI/HpaII Linker 2CGTTCTAGACTCATC100 µM100 µl
      TRIS/Cl pH 81 M2 µl
      NaCl5 M2 µl
      EDTA0.5 M0.4 µl
    • prepare EcoRI linker/adaptators for 200 reactions:
    • destilled sterile water175.5 µl
      EcoRI Linker 1CTCGTAGACTGCGTACC100 µM10 µl
      EcoRI Linker 2AATTGGTACGCAGTCTAC100 µM10 µl
      TRIS/Cl pH 81 M2 µl
      NaCl5 M2 µl
      EDTA0.5 M0.4 µl
    • heat 10 min to 65°C, cool slowly to room temperature
    • prepare ligation master mix:
    •  1x96x (2 enzymes)144x (3 enzymes)
      T4 ligase NEB400 U/µl0.5 µl (200 U)53 µl79 µl
      ligation buffer10x1 µl106 µl158 µl
      EcoRI Linker5 µM1 µl106 µl158 µl
      AciI/HpaII Linker50 µM1 µl106 µl158 µl
      ATP10 mM2 µl192 µl317 µl
      sterile destilled water4.5 µl475 µl648 µl
    • mix well
    • inactivate digestion by incubation at 65°C for 20 min
    • add 10 µl of ligation master mix to each well, mix by pipetting
    • seal with aluminum sheet, centrifuge shortly
    • incubate at 37°C for 2 to 3 hours
    • prepare PCR master mix:
    •  1x96x (2 enzymes)144x (3 enzymes)
      Taq 10x bufferhigh efficiency2 µl211 µl317 µl
      Taq polymerase0.5 U/µl2 µl211 µl317 µl
      dNTPs10 mM each2 µl211 µl317 µl
      primer AciI/HpaII
      GATGAGTCTAGAACGGTCC
      10 µM2 µl211 µl317 µl
      fluorescent primer EcoRI
      GACTGCGTACCAATTCCTG
      0.5 µM2 µl211 µl317 µl
    • mix well
    • distribute 10 µl to one or two 96 well PCR plates
    • dilute 10 µl of digestion/ligation reaction with 190 µl sterile destilled water (1:20)
    • Mix well by pipetting through about 100 µl pipettes
    • add 10 µl of this diluted DNA to the 10 µl PCR master mix
    • or:
    • add 10 µl of undiluted DNA to the PCR master mix and use PCR conditions II
    • seal with aluminum sheet, centrifuge shortly
    • PCR:
    • 96°C30 sec
      14 cycles:
        | 96°C30 sec
        | 65°C30 sec
        | decrease temperature by 0.7°C every cycle
        | 72°C1 min
      25 cycles (15 cycles for PCR condition II):
        | 96°C30 sec
        | 56°C30 sec
        | 72°C1 min
      60°C30 min
      4°Cforever
     
  • 2nd day:
    • prepare master mix for CEQ-8000 capillary sequencer:
      1x96x (2 enzymes)144x (3 enzymes)
      SLS40 µl4032 µl6336 µl
      600 bp size marker0.1875 µl20 µl29 µl
    • distribute 40 µl master mix into 96 well plates
    • add 1 µl of MS-AFLP PCR to each well
    • add one drop of mineral oil to each well
    • seal with aluminum sheet, centrifuge shortly
    • run on CEQ-8000
    • call binary data

Go back