New digs

OQx70jjBSLOMI5ackhxm_urbex-ppc-030I’m moving the site from my own hand-coded html over to an instance of WordPress for 2015.  There are several reasons why I am doing this:

  1. I don’t seem to update too often if I hand code it making my lab webpage rather stale.
  2. Syndication and federation of posts can add dynamic content from lab members to the site.
  3. I’m moving towards this approach in teaching as well so I thought I would eat my own dogfood.

We will see how it goes.  Lots of stuff to add and go over.

Diluting Primers

Diluting Primers

To dilute primers for PCR, you need to first make a 100µM stock solution.  To accomplish this, you find the number of nMol on the tube and add 10X that much water in µl.  This is your stock solution.  Then for your working solution, dilute it to 10µM and work with that one.

Restriction Enzymes for AFLP’s

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:

  1. Order from NEB (http://neb.com), they are the shiznit (n.b., that is a technical term).
  2. 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.
  3. 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).
  4. Template DNA should be no more than 300 ng.  We DO NOT need a lot of template to start with.