扩增酶切片段多态性（AFLP Protocol）

Restriction digestion Master mix preparation: Prepare a master mix of the following per sample, plus 5 to 10% extra to allow for pipetting loss. 5X R/L buffer (see page 4 for recipe) 6.0 μl EcoR I (12 U @20 U/μl) 0.6 μl Mse I (8 U @ 4 U/μl) 2.0 μl Water to 10 μl Reaction time: Incubate for at least 1 hr. at 37°C, but not longer than 3 hrs, before proceeding with the ligation.

Adapter ligation Master mix preparation If 10 μl of the restriction reaction was removed for gel analysis, prepare a ligation master mix of the following, per sample, plus enough for 5-10 extra samples: EcoRI adapter (@ 5 pMol/μl) 0.5 μl MseI adapter (@ 50 pMol/μl) 0.5 μl ATP (10 mM, pH 8.0) 0.5 μl 5X R/L buffer 1.0 μl sdH2O 2.0 μl T4 DNA ligase (0.5 Weiss U @ 1 U/μl) 0.5 μl TOTAL 5.0 μl Reaction time: Incubate for at least 3 hrs. (preferably overnight) @ 37° C. After incubation, dilute each R/L mix 1:10 with sdH 2 O.

Preamplification Master mix preparation Prepare a master mix with the following amounts per sample, plus 5-10 samples extra: 10X PCR buffer 3.0 μl dNTP mixture (2.5 mM ea.) 2.4 μl E primer (@ 50 ng/μl ≅ 8.3μM) 1.0 μl M primer (@ 50 ng/μl ≅ 8.3μM) 1.0 μl Taq polymerase (@ 5 U/μl) 0.4 μl sdH 2 O 19.2 μl μl per sample 27.0 μl

Important : If pre-amplifications are to be done in 96-well PVC plates instead of polycarbonate or polypropylene plates or tubes, it may be necessary to add 2.0 μl of 10 μg/μl BSA per sample to the Taq-buffer mix, and decrease the amount of H2O to 5.76 μl per sample. DO NOT use a hot start PCR protocol, or a hot start polymerase (e.g. AmpliTaq Gold), for the pre-amplification! The adapters are non-phosphorylated, so only the top strand is ligated. The bottom strand of the adapter will separate from the rest of the template first and must be re-synthesized during the initial heating stage. This requires polymerase activity during the initial heating. PCR program Place reactions in the thermocycler, and run the following PCR amplification profile: 28 cycles: 15 sec @ 94°C denaturation 30 sec @ 60°C annealing 60 sec + 1sec/cycle @ 72°C extension 1 cycle: 2 min @ 72°C final extension hold: 4°C

Final Amplification Master mix preparation (single dye reactions) For each E/M primer combination to be used, prepare the following master mix, per sample, in an Eppendorf tube, plus enough for 5-10 samples extra: 10X PCR buffer 2.0 μl dNTP mixture (2.5 mM ea) 1.6 μl IRD-labeled E-primer (@ 6 ng/μl ≅ 1μM) 0.83 μl M-primer (@ 50 ng/μl ≅ 8.3μM) 0.6 μl Taq polymerase (@ 5U/μl) 0.24 μl dH 2 O 9.73 μl Final volume 15.0 μl