AFLP Setup

This protocol, currently (occasionally) used in the Wolf lab, has been adapted (by Paul Wolf and Mark Ellis) from several sources: The AFLP plant mapping guide from applied Biosystems, the AFLP protocols of Laboratoire de “Biologie des Populations d’altitude” (Grenoble. France), and the KeyGene protocols by Matt Gitzendanner (University of Florida). The original AFLP protocol was that of Vos et al. 1995 Nucleic Acids Research 23:4407-4414. Restriction and Ligation Reactions

The first step in preparing your samples for analysis is to cur the genomic DNA with two restriction enzymes (typically EcoRI and Msel). You will then ligate the adapters to the overhanging sticky end produced by the restriction enzymes.

There are two protocols described below; one protocol calls for the restriction and ligation reactions to occur in the same tube at the same time, and the other protocol requires that these two steps be done separately. Note: the restriction enzyme Tru91 is an isoschizimer (same recognition sequence and em pattern) of Msel and is considerably cheaper. We have found that substituting Tru91 for Msel does not effect the results. Thus, while the protocol still references the commonly used Msel enzyme we typically use Tru91 for our studies.

AFLP Oligonucleotide Information

ADAPTORS:

Eco-F: 5′-CTC GTA GAC TGC GTA CC-3′

Eco-R: 5′-AAT TGG TAC GCA GTC TAC-3 ‘

Mse-F: 5′-GAC GAT GAG TCC TGA G-3′

Mse-R: 5′-TAC TCA GGA CTC AT-3 ‘

PRIMERS:

We have historically just used A’s as the + I nucleotides for 1+ I reactions. For 3+3 reactions, we generally use “A” plus some combination of nucleotides that gives us a total of 3 G’s or C’s on both primers.

MSE-NNN: 5′-GAT GAG TCC TGA GTA ANN N-3 ‘ •••NOTE THE TERMINAL N

ECO-NNN: 5′-GAC TGC GTA CCA ATT CNN N-3 ‘ •••NOTE THE TERMINAL N

Getting the Adapters Ready Note: this only needs to be done the first time that you mix the adapter pairs. For each enzyme used, there is an adapter pair that will be ligated to the sticky ends. The adapters come as single strands, so the two strands of each adapter must be annealed to each other before they can be used.

For EcoRI Adapter pair (final conc of 5 micromolar), mix:

For Msel Adapter pair (final conc of 50 micromolar), mix:

After mixing the adapters

Heat at 95°C for 5 min to denature. Then allow to cool slowly in a Styrofoam box to renature completely.

Setting Up the Reactions For the restriction and ligation reactions, two master mixes are made up: one the enzymes, and one containing the adapters. Use the reaction setup sheets at the end of this protocol to set up the reactions. Here are some general comments regarding the restriction/ligation reactions:

Below are two recipes for restriction-ligation: the first is the two-step procedure that uses the AFLP core kit from Life Technologies. This is followed by the one-step that we will follow subsequently when we buy the reagents separately.

Kit Recipe First mix all the ingredients (minus DNA) enough for all tubes plus three extra (pipetting error). Aliquot the mix, then add the DNA last, changing tips to avoid contamination across reactions.

Step 1. Digestion (per reaction tube):

Incubate 2 hours @ 37 C, then 15 min at 70 C to inactivate the enzymes.

Step 2. Ligation. Add:

For a total now of 2 micoliters per tube.

Incubate @ 18-22 C (room temp) for a further 2 hours, then store @ 4 C.

Dilute 10-fold with distilled water. Normal Protocol

(Once we exhaust the kit): In a 0.2 micoliter tube place the following (you can make a master mix and aliquot later into the ligation tubes):

Enzyme master mix:

Total per tube = 1 microliter

Then prepare ligation tubes(all except template DNA can be made as a master mix):

Total: 5.5 micoliters

Add template DNA 5.5 micoliters (at about 10ng/micoliter).

Mix well, centrifuge briefly to spin down droplets, and incubate at 37 C for 2 hours. Store at 4 C.

Add 90 micoliters TE0.1 to each tube (Note that the EDTA in the TE is at 0.1 M).