Professor John Paul
Lab 8 Entry
Plant DNA Extraction
On October 17th, I arrived at the lab and began our protocol for the plant DNA extraction. I started by obtaining three random capsules with different plant tissues and silicon pellets in each of them from my professor. I then labeled three 2.0 ml tubes with my initials and the plant sample code that was given on the capsule of each of the three plant variations (JP1231, JP1298, and JP1229). Three small sterile 3.2-mm stainless steel beads were added into each of the newly labeled 2.0 ml tubes. The size of about a fingernail of each of the three plant samples was then transferred with a sterile pair of forceps into the correct corresponding 2.0 ml tubes that were labeled with the sample code, along with the steel beads. Between each plant sample transfer, I wiped the forceps with 70% ethanol to ensure no cross contamination was occurring.
My lab group and I then brought each of our three 2.0 ml tubes over to our professor in the corner of the lab, where he showed us how to assemble the modified reciprocating saw in order to crush the plant tissue with the steel beads during the vibrations. I then took my three tubes over to the centrifuge and had them spun for about 15 seconds at a fast speed. Next, I brought my tubes back to the table and added 434 microliters of preheated grind buffer into each tube using a p1000 micropipette and filtered tips and then brought these tubes to a hot water bath to be incubated for ten minutes total and inverting the tubes every three minutes to mix the contents inside.
After the ten minutes passed, I brought the tubes back to my table and added 130 microliters of 3M ph 4.7 potassium acetate into each tube with a micropipette and filtered tips. Then I inverted the tubes and placed them in the centrifuge to be spun at maximum force for 20 minutes, making sure my lab group balanced out our tubes in the centrifuge. I then brought these tubes back to our table and acquired three new 1.5 ml tubes from our professor and labeled them with our initials and plant sample codes. I then used micropipettes and filtered tips and transferred out only the supernatant (avoiding any precipitate from inside the tube) from the 2.0 ml tubes that I just took out of the centrifuge and put them into the correct corresponding 1.5 lm tubes that we just got. Since each of my supernatant levels for each plant sample code measured out to 400 microliters, I then added 600 microliters of binding buffer to each of the 1.5 ml tubes (I added 1.5 volumes of the binding buffer of my total measurement of supernatant that I acquired).
After that, I got three new Epoch spin column tubes, initialed and labeled each with the three different sample codes. I transferred 650 microliters of each of the mixtures from the 1.5 ml tubes into the three new and correct corresponding Epoch spin column tubes. I then centrifuged these tubes for ten minutes and removed the waste liquid that settled to the bottom of the tubes into a waste beaker. I then put the remaining mixtures from the 1.5 ml tubes and put them into the same corresponding Epoch spin column tubes to centrifuge again for ten minutes and emptied out the waste liquid that settled at the bottom. I added 500 microliters of 70% ethanol to each of the Epoch spin column tubes and centrifuged them for eight minutes. After the eight minutes, the waste liquid at the bottom was removed into the waste beaker. I then once again added 500 microliters of the same ethanol to the Epoch tubes and centrifuged them for eight minutes once more to collect and remove the waste liquid. Then I removed the “flow-through” parts of the Epoch tubes and kept the “column” parts.
Once this was done, I took my three “columns” to the centrifuge and spun them for five minutes to remove any residual ethanol from the previous steps. The “columns” were then placed into three newly initialed, sample coded, and dated 1.5 ml centrifuge tubes. I added 100 microliters of preheated pure sterile water into each of these 1.5 ml tubes and let them sit for five minutes. Then I centrifuged the tubes for two minutes at high spin to elute the DNA. The tubes holding our plant DNA was then given to our professor.
Thoughts: I hope our professor lets me use the modified reciprocating saw rack for later experiments.