Que sera sera

This week, I reviewed my latest protocol in order to locate all of the chemicals required, most of which were already on hand in the lab. There were only two things on my "shopping list" that needed to be ordered: sarkosyl and proteinase k.

Sarkosyl is a surfactant (read detergent). Surfactants work on the basis of lowering the surface tension of liquids they are introduced to. For example oil and water do not "mix," with the introduction of the proper surfactant they could be mixed. Sarkosyl's role in DNA extraction is in the disruption of membranes that is, it renders the proteins within the cell membrane soluble, thereby assisting in lysing the cell.

Proteinase k is an enzyme (hence the "ase" part of its name) that acts upon proteins (the protein part of its name). It will essentially digest proteins that contaminate the extraction results. It will also act upon any nucleases (that are present naturally) and negate their breaking of nucleotide chains (DNA strands).

So the order was made and I'm just waiting on these two items to come in and then I can create my reagents and off I go.

or so I thought...

Upon detailed examination of my protocol (an attempt to fully understand every step in process to make it easier when I actually conduct the extraction), I noted this:

• SDS/Sarkosyl lysis buffer
3 volumes of 4% SDS buffer
1 volume of 5% Sarkosyl Buffer
5 μg/mL proteinase K
0.12% of β-mercaptoethanol
Add 200 μL of Proteinase K (2Omg/mL) and 100 μL of β-mercaptoethanol to 60 mL of 4% SDS buffer, and 20 mL of 5% sarkosyl buffer.
Do not refrigerate or autoclave. 

It appears as though there is some conflicting information (high-lited in blue). The first 4 lines of the quote above are like the ingredients of the buffer, and the sentence beginning with add is like the recipe of how to make it. Five micrograms per milliliter and twenty milligrams per milliliter are vastly different concentrations! I could understand if the first concentration given was higher than the other - that would indicate that I would need to make a dilution of it in order to conform to the "recipe." How exactly would I get from a lower concentration to a higher though? I consulted with Josh and Matt and they were also unable to make sense of this either. Our consensus was for me to find other protocols that use proteinase k and compare concentrations.

Just when I thought I was done digging through protocols... que sera sera...

To date, I haven't delved into this fully. It does appear that 20mg/mL is a common concentration for proteinase k. I will look into this more as I want to be certain so the potential for error is reduced.