Still plugging along

This week I conducted another run with the phenol chloroform extraction. In this attempt, I halved the amount of initial plant tissue, and worked to reduce the break points in the protocol. This being my second usage of the protocol coupled with my conducting it on a day in which I had more hours to devote to it, allowed me to conduct the protocol seamlessly with no breaks other than incubation time. Unfortunately the results still exhibited signs of shearing.

This is a picture of the gel from the camera mounted on the UV light box. The beginings of banding can be seen, but the DNA sample is sheared (the lanes are just streaks)

I am currently working on two other ideas that will hopefully reduce the shearing. The first step of the protocol is to grind the leaf tissue samples (-80° C) in the lysis buffer containing the 5% sarkosyl solution and the 4% SDS solution, and then incubate it at 50° C for two hours. It is my suspicion that shearing may be occurring in the grinding. I have taken tissue samples and incubated them in the buffer for an extended period of time without grinding. I am not sure if this will yield anything, but it does merit testing. The other potential shearing point that I see is the pipetting of the samples. I suspect that drawing the samples through the narrow tip of a standard pipette could cause shearing. Since there are not any wide bore pipettes available for use, I will snip the pipette tips back to a wider opening for each pipetting. I had intended on this today, but unfortunately the BIO 181 classes are using the fume hood today. Normally Tuesday and Thursday are the days I have enough time in the lab to carry out the full extraction. Next Tuesday is a holiday, so I will not be able to revisit this until Thursday. Hopefully I will see a clean nicely banded lane in the well

As to the Sucrose extraction that Matt clued me in on –

I have read that this is to induce an osmotic shock within the cells as a means of cell lysis. The addition of the sucrose in the solution increases the solute concentration outside of the cell. This rapid change of concentration results in an equally rapid change in the movement of water across the membranes (osmosis). The drastic change in pressures should lyse the cell. If this is actually what happens, I should be able to use a sucrose lysis buffer and not have to manually grind the tissues. This is in the same vein of what I am doing with the current extraction – attempting to lyse cells with out physical tissue homogenization. There was one protocol I read that specifically stated that the sucrose buffer was very effective for plants that had been previously shown to be difficult to extract DNA from. Unfortunately they didn’t say why. I am still digging to see if I can find more information on this.