again, and again, and again, again...

Again, I will be starting over... I feel as though I have said that before. Early on this week I made the decision to retry the higher quality extraction with the wizard kit I used at the end of last semester. There was one slight hitch. The extraction buffer I used is no longer viable. Embarrassingly enough,  as I recall, the buffer I used last semester was just given to me, and I had no idea of its composition or who made it. Much of this week was then spent researching different extraction methods and buffers that would work for me. MANY of these call for the leaf sample to be frozen in liquid nitrogen and then ground up before adding buffers - not really an option for me. Also of note many methods require the use of phenol-chloroform (two nasty chemicals). I wasn't told not to use phenol-chloroform, but it felt as if it were implied that another method may be better.

After reviewing a rather large amount of scientific literature (predominately journals), I did find a method that didn't require the above. Unfortunately not all of the reagents required for the buffer were on hand, but we anticipate delivery early next week. The components of the buffer are:

200 mM Tris HCl pH 7.5 This maintains a stable pH in solution as cells are lysed.

250 mM NaCl Provides Na+ Ions that bond to the negatively charged phosphate groups that are part of DNA (if the Na+ didn't do this the molecules would repel each other)

25 mM EDTA Is a chelating agent that neutralizes metal ions like Mg2+ and CA2+ which are needed by DNA polymerase and other enzymes. Doing so essentially stops the action of these enzymes and preserves the DNA in it's intact state.

0.5% SDS is a detergent.

 

I can't wait to finally get some results! The end of the semester is approaching much faster than I care to admit! 

For general information good results would look something like this:

Image from Promega's website (the maker of the wizard kit.