Purpose:

The purpose of this lab was to observe the cells and extract the DNA of our sample. As a result of this lab we will be able to perform PCR/Gel Electrophoresis next time. Our group had to observe our cells under a compound microscope because we did not go to open lab last week.

Methods:

Counting Cells

• Obtain 2ouL of Iodine and 20uL of cells and place it in a micro centrifuge tube and vortex.
• Place five 2uL drops of the mixed sample and observe under a compound microscope.
• If cells are difficult to locate, dilute with the sample.

E.Z.N.A. Tissue DNA Protocol

• Prior to starting the protocol: Set a heat block to 7o˚C, Heat Elution Buffer to 70˚C in the heat block, and chill PBS to 4˚C.
• Prepare the cell suspension: Prior to the protocol thaw the frozen cell samples and wash the cells with cold PBS.
  • Resuspend the cells in 200uL of fresh PBS
  • Add 25uL OB Protease solution
  • Use the vortex to mix the solution
• Add 22ouL of BL Buffer. This will destabilize hydrogen bonds and provide optimal conditions for the DNA transfer to the silica.
• Incubate at 70˚C for 10 minutes in the heat block. During this process vortex the tube once.
• Add 200uL of 100% ethanol. Then vortex to mix thoroughly. Be sure to measure this carefully.
• Insert the HiBind. DNA Mini Column into a 2mL collection tube.
• Transfer the entire sample from heat block to the HiBind.
• Place this in the centrifuge this at 10,000xg for 60 seconds.
• At this point discard the filtrate and reuse the collection tube.
• Add 500uL HBC Buffer to the column. This will remove the impurities from the column while leaving the DNA bound to the silica.
• Centrifuge this sample at maximum speed for 30 seconds.
• Discard the filtrate and collection tube.
• Insert the HiBind DNA Mini Column into a new 2mL collection tube.
• Add 700uL DNA wash buffer and centrifuge at maximum speed for 30 seconds. Discard the filtrate and reuse the collection tube.
• Repeat the steps for a second DNA Wash Buffer wash step. This will help remove the salt impurities.
• Centrifuge the empty HiBind DNA Mini Column at maximum speed for 2 minutes to dry the column.
• Transfer the HiBind DNA Mini Column into a nuclease free 1.5mL microcentrifuge tube. Label the tube with your groups name.
• Add 100uL Elution Buffer heated to 70˚C. Let it sit at room temperature for 2 minutes.
• Centrifuge at maximum speed for 1 minute and store the eluted DNA at -20˚C.

Results:

The results were the DNA we extracted from the cells. We will be performing Gel Electrophoresis on the DNA next week. Regarding the cell counts from our original sample I viewed counts of 56, 26, and 15 cells in the three drops I viewed. The other two drops were unable to be counted. There counts seemed high and were a very rough estimate. I cannot guarantee the accuracy of these results.

Conclusion:

Next week we will be performing Gel Electrophoresis and actually extracting the DNA. This was my first time performing an actual protocol with some degree of difficulty. I found it to be very rewarding and fascinating how all these steps work together to get a specific final product. It was also very interesting to understand how each ingredient had its own purpose in extracting the DNA.