The key to the AFLP protocol is to be able to digest two restriction enzymes (RE’s) simultaneously and be able to ligate onto these sticky ends primers of known concentration. When you purchase new primers, you need to aliquot out usable volumes because repeated freeze/thaw cycles reduce RE efficiency. Here are some guidelines:
- Order from NEB (http://neb.com), they are the shiznit (n.b., that is a technical term).
- Do not order the MOST CONCENTRATED but be reasonable. We typically do not work in the volume of needing 100,000 U/ml, be reasonable.
- When you receive the shipment from NEB, aliquot and dilute such that we get 5 U in 20µl putting in 2µl stock (e.g., shoot for 2.5 U/µl).
- Template DNA should be no more than 300 ng. We DO NOT need a lot of template to start with.