Potato Germplasm Enhancement Laboratory

Obihiro Univerisity of Agriculture and Veterinary Medicine
West 2-11, Inada, Obihiro, Hokkaido 080-8555, Japan

AFLP (double digestion and ligation in one tube)

1. Double digestion and ligation

ComponentVolumeReaction mix (xn)
DNA + sterile water4 μl (100 ng)-
10x NEBuffer 4 (NEB)2 μl (1x)2 x n μl
EcoRI (20 units/μl, NEB)0.125 μl (2.5 units)0.125 x n μl
MseI (50 units/μl, NEB)0.05 μl (2.5 units)0.05 x n μl
T4 DNA ligase (3.2 Weiss units/μl, NEB)0.156 μl (0.5 units)0.156 x n μl
100 mM ATP (Roche)0.2 μl (1 mM)0.2 x n μl
25 μM MseI adapter0.5 μl (12.5 pmoles)0.5 x n μl
5 μM EcoRI adapter0.25 μl (1.25 pmoles)0.25 x n μl
Sterile water12.719 μl12.719 x n μl
Total20 μl16 x n μl

Note: T4 DNA ligase (NEB, M0202S, 400,000 units/ml = 3.2 Weiss units/μl)
One Cohesive End Ligation Unit = 0.008 Weiss Unit

  1. Prepare 100 ng of DNA sample (25 ng/μl) in a volume of 4 μl in a 1.5 ml tube.

  2. Add 16 μl of Reaction mix.

  3. Mix well by finger-tapping and spin (no vortex), and then, incubate overnight at 37°C.

  4. Add 30 μl of sterile water.

2. Pre-amplification

ComponentMix aMix bx No. of samples
Mix AMix B
Diluted DNA1 μl---
2x Ampdirect Plus5 μl2.5 μl5 x n μl2.5 x n μl
BIOTAQ HS DNA Polymerase (5 units/μl)-0.05 μl-0.05 x n μl
3 μM pre-amp E (5'-GACTGCGTACCAATTCA-3')1.5 μl-1.5 x n μl-
3 μM pre-amp M (5'-ATGAGTCCTGAGTAAC-3')
or 3 μM pre-amp MG(5'-ATGAGTCCTGAGTAAG-3')
1.5 μl-1.5 x n μl-
Sterile water1 μl2.45 μl1 x n μl2.45 x n μl
Total10 μl5 μl9 x n μl5 x n μl

  1. Heat 'Mix B' at 95°C for 10 min in a heat block.

  2. Mix 'Mix A' and 'Mix B' together.

  3. Place 1 μl of diluted DNA into the bottom of PCR tube, and then, dispense 14 μl each of the above mixture.

  4. PCR - 72°C 2 min, 95°C 3 min, 20 cycles (95°C 30 sec, 60°C 1 min, 72°C 1 min), 72°C 5 min, soak at 4°C)

  5. Measure DNA concentrations (normally 150-250 ng/μl).

  6. Pick 10 μl of PCR products and add sterile water (normally 290-450 μl) to adjust DNA concentration to 5 ng/μl.

3. Selective amplification

ComponentVolumeReaction mix (xn)
Pre-amplified DNA (5 ng/μl)2 μl-
2x Ampdirect Plus5 μl (1x)5 x n μl
BIOTAQ HS DNA Polymerase (5 units/μl)0.05 μl (0.25 units)0.25 x n μl
3 μM E-primer (5'-GACTGCGTACCAATTCANN-3')1 μl (0.3 μM)1 x n μl
or 3 μM MG-primer (5'-GATGAGTCCTGAGTAAGNN-3')
1 μl (0.3 μM)1 x n μl
Sterile water0.95 μl0.95 x n μl
Total10 μl8 x n μl

  1. Add 2 μl of pre-amplified DNA (5 ng/μl) and dispense 8 μl of Reaction mix.

  2. PCR as usual for AFLP (95°C 10 min, 65°C 30 sec, 72°C 1 min, then the annealing temperature is subsequently reduced each cycle by 0.7°C for the next 12 cycles, and is continued at 56°C for the remaining 23 cycles. Final addition of 72°C 5 min, then soak at 4°C)

  3. Check 5 μl on a 2.0% agarose gel. If the results are OK, add 5 μl of Formamide dye into the remaining sample for polyacrylamide gel electrophoresis.

How to make

25 μM MseI adapter

50 μM AFLP- MseI-1 (5'-GACGATGAGTCCTGAG-3')90 μl
50 μM AFLP- MseI-2 (5'-TACTCAGGACTCAT-3')90 μl

  1. Mix together in a 1.5 ml tube.

  2. Heat in a heat block at 95°C for 5 min, then turn off and leave for several hours to cool slowly.

5 μM EcoRI adapter

50 μM AFLP- EcoRI-1 (5'-CTCGTAGACTGCGTACC-3')50 μl
Sterile water400 μl

  1. Mix together in a 1.5 ml tube.

  2. Heat in a heat block at 95°C for 5 min, then turn off and leave for several hours to cool slowly.