DATA & PROTOCOLS » AFLP PROTOCOL

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.

+1 PCR Reactions (Preselective PCR)

The first PCR reaction uses primers that match the adapter sequence and have one additional '"selective" base. This reduces the number of >bands that will be amplified. Theoretically this selective base could be any base, we use an A on the EcoR1 primer (EcoR1A) and a C on the Msel primer (Mse1 + 1C). The T4 DNA ligase only ligates one of the strands of the adapter to the fragment. The other is held on by base-pair binding to the other adapter strand. Thus, the first step of the+ 1 PCR reaction is a 72 C hold that allows the Taq polymerase to ligate the other strand.

If you perform a Hot-Start, or place the +1 reactions into a hot thermal-cycler, or omit the initial 72 C hold, you will loose the second strand and the PCR reaction won't work. Do not use AmpliTaq Gold for +1 reactions as the Taq pol will not be active in the initial 72 C hold.

Use the set-up sheet at the end of this protocol to set-up the +1 reactions.

It is imperative that all solutions are thawed completely and mixed well when setting up the reactions.

In a 0.2 micoliter tube, (make a large mix as usual):

Reagent Stock Conc. Final Conc. Vol. (micoliters) Vol. x N (fill in)
Taq buffer 10X 1X 2.5  
Taq DNA pol 5 units/micoliter 0.25 units 0.1  
MgCl2 50 mM (check) 1.5 mM 0.75  
dNTPs 5 mM 0.12 mM 0.6  
Primer-Mse-C 10 micomolar 0.2 micromolar 0.5  
Primer Eco_A 10 micomolar 0.2 micromolar 0.5  
Diluted dig-lig     3  
dd H2O        
Total     25  

Use the following PCR parameters: 72 C for 2min: then 30 time (94 C for 30 sec: 56 C for 30 sec: 72 C for 2 min): then 60 C for 10 min, then hold at 4 C. This profile is on the PE GeneAmp 2400. Run 10 microliters of the +1 reaction on a 1.5% agarose gel. You should see a smear in the 100 to 1000 bp range. Sometimes bands are visible through the smear.

Add 100 micoliters TE0.1 to the remaining reaction.

+3 PCR Reactions (Selctive PCR)

For the final PCR reaction, the +1 products are used as template, and the primers are the same sequence except that they have two additional selective bases. The EcoR1+3 primers are labeled with a fluorescent dye (6-FAM in the Wolf lab) that can be detected by the ABl 377.

Initially, you should test all EcoR1+3/Mse1+3 primer combinations individually to identify the ones that work best (give the most readable and numerous polymorphic bands). If there are several EcoR1+1primers that work well wilh one Mse1+3 primer, and they are labeled with different dyes, then you can test them in multiplex reactions (where multiple EcoR1+3 primers are added to a reaction with a single Mse1+3 primer) to verify that they produce the same bands as when they are run individually. If muhiplexing does not work, it may still be possible to run separate PCR reactions, combine the product, and run the samples in a single lane. However, as less of each reaction is loaded, the signal strength is reduced.

It is imperative that all solutions are thawed completely and mixed well when setting up the reactions. This is especially important the the labeled primers as they tend to settle out of solution faster than regular primers.

Reagent Stock Conc. Final Conc. Vol. (micoliters) Vol. x N (fill in)
Taq buffer 10X 1X 1.25  
AmpliTaq gold 5 units/micoliter 0.25 units 0.1  
MgCl2 50 mM (check) 2 mM 0.5  
dNTPs 5 mM 0.12 mM 0.3  
Primer-Mse-C** 10 micomolar 0.2 micromolar 0.25  
Primer Eco_AGG 10 micomolar 0.04 micromolar 0.05  
BSA 1 mg/mL 8 micrograms/mL 0.1  
Diluted +1 rxn     2.5  
dd H2O        
Total     12.5  

Use the following PCR parameters: 94 C 2 min: 12 x (94 C 30 sec; 65 C 30 sec [reduce by 0.7C each cycle]; 72 C 2 min) then 24 x (94 C 30 sec; 56 C 30 sec; 72 C 2 min) then 72 C 10 min, then hold at 4C. This profile is on a PE 9700.

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