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.
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.
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 ‘
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
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:
25 micoliters of EcoRI Forward Adapter (@ 100 micromolar)
25 micoliters of EcoRI Reverse Adapter (@ 100 micromolar)
450 micoliters of TE
500 micoliters total
For Msel Adapter pair (final conc of 50 micromolar), mix:
250 micoliters of Msel Forward Adapter (@ 100 micromolar)
250 micoliters of Msel Reverse Adapter (@ 100 micromolar)
500 micoliters total
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.
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:
Keep everything on ice as much as possible, to keep the enzymes from working before you want them to, minimize evaporation, and keep the adapters renatured.
Make sure you completely thaw and mix all buffers, especially the T4 DNA ligase buffer. Because the T4 ligase buffer has ATP, which is rather unstable, the stock tube of this reagent should be aliquoted it into small tubes to reduce freeze/thaw cycles.
A note on unit definitions of T4 ligase: there are actually two unit definitions for T4 ligase: Weiss units and cohesive-end ligation units. These are very different! In fact, 1 cohesive-end ligation unit (definition used by New England Biolabs) is only 0.015 Weiss units (definition by just about everyone else). The thing is that the definition of a Weiss units is such that 0.01 Weiss units will ligate 1 microgram of DNA min, so that’s about all you need in your reactions. In general, 1 cohesive-end ligation unit (or 0.0 15 Weiss units) per reaction is good.
The T4 Ligase is really concentrated, so you don’ t need much and it is hard to pipette accurately. Don’ t worry about this, as long as you get some in I think you’ re fine.
Since the reaction volumes are small, and any evaporation could cause EcoR1 to cut randomly (EcoRI* activity), in addition to cutting at specific sites, set up the reaction in 200 microliter tubes and use a thermocycler with a hot top, or add mineral oil for a conventional thermocycler.
For the protocol that combines the restriction and ligation procedures, any fragment-fragment re-ligations that are made will be recut by the restriction enzymes. The adapter sequence is such that the restriction site is not regenerated following fragment-adapter ligation.
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.
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):
2.5 micoliters reaction buffer
1 micoliter EcoR 1/Mse1 mix
50 ng template DNA
Distilled water to a total volume of 12.5 micoliters
Incubate 2 hours @ 37 C, then 15 min at 70 C to inactivate the enzymes.
Step 2. Ligation. Add:
12 micoliters adaptor/ligation solution
0.5 micoliters T4 DNA ligase
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.
(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:
T4 buffer 10X
BSA @ I mg/ml
Mse1: I unit
EcoR1: 5 units
T4 ligase: 1 unit
Total per tube = 1 microliter
Then prepare ligation tubes (all except template DNA can be made as a master mix):
T4 buffer 10X: 10 micoliters
NaCl 0.5M 1.0 micoliter
BSA @ 1 mg/ml 0.5 micoliter
Enzyme master mix 1.0 micoliter
Mse adapter 1.0 micoliter
EcoR1 adapter 1.0 micoliter
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).