AFLP (double digestion and ligation in one tube)
1. Double digestion and ligation
| Component | Volume | Reaction mix (xn) |
|---|---|---|
| DNA + sterile water | 4 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 adapter | 0.5 μl (12.5 pmoles) | 0.5 x n μl |
| 5 μM EcoRI adapter | 0.25 μl (1.25 pmoles) | 0.25 x n μl |
| Sterile water | 12.719 μl | 12.719 x n μl |
| Total | 20 μl | 16 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
- Prepare 100 ng of DNA sample (25 ng/μl) in a volume of 4 μl in a 1.5 ml tube.
- Add 16 μl of Reaction mix.
- Mix well by finger-tapping and spin (no vortex), and then, incubate overnight at 37°C.
- Add 30 μl of sterile water.
2. Pre-amplification
| Component | Mix a | Mix b | x No. of samples | |
|---|---|---|---|---|
| Mix A | Mix B | |||
| Diluted DNA | 1 μl | - | - | - |
| 2x Ampdirect Plus | 5 μl | 2.5 μl | 5 x n μl | 2.5 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 water | 1 μl | 2.45 μl | 1 x n μl | 2.45 x n μl |
| Total | 10 μl | 5 μl | 9 x n μl | 5 x n μl |
- Heat 'Mix B' at 95°C for 10 min in a heat block.
- Mix 'Mix A' and 'Mix B' together.
- Place 1 μl of diluted DNA into the bottom of PCR tube, and then, dispense 14 μl each of the above mixture.
- 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)
- Measure DNA concentrations (normally 150-250 ng/μl).
- 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
| Component | Volume | Reaction mix (xn) |
|---|---|---|
| Pre-amplified DNA (5 ng/μl) | 2 μl | - |
| 2x Ampdirect Plus | 5 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 |
| 3 μM M-primer (5'-GATGAGTCCTGAGTAANNN-3') or 3 μM MG-primer (5'-GATGAGTCCTGAGTAAGNN-3') | 1 μl (0.3 μM) | 1 x n μl |
| Sterile water | 0.95 μl | 0.95 x n μl |
| Total | 10 μl | 8 x n μl |
- Add 2 μl of pre-amplified DNA (5 ng/μl) and dispense 8 μl of Reaction mix.
- 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)
- 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 |
- Mix together in a 1.5 ml tube.
- 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 |
| 50 μM AFLP- EcoRI-2 (5'-AATTGGTACGCAGTCTAC-3') | 50 μl |
| Sterile water | 400 μl |
- Mix together in a 1.5 ml tube.
- Heat in a heat block at 95°C for 5 min, then turn off and leave for several hours to cool slowly.