PURPOSE: The purpose of today’s lab is to clean the cells, lyse the cells to extract the DNA, and then purify the DNA. We do this so that we can the perform PCR next class on the DNA

PROCEDURE:

Pre-Protocol:

• set heat block to 70 degrees C heat elution buffer
• Chill the PBS to 4 degrees C by placing the tubes in a bucket of ice

Pellet/wash the cells:

*no one from the group came in during open lab so we must start by pelleting our cells for the first time.

1. Spin cell solution for 5 mins at 3000 x g
2. remove supernatant with a p1000 being careful to not disturb the pellet
3. Resuspend (wash cells) with 1 ml of cold PBS per test tube
4. Spin 5 min at 3000 x g
5. Once again, remove supernatant using p1000 being careful not to disturb the pellet
6. Resuspend one more time however this time, add 100 ul cold PBS per tube, mix the content, combine the content from both test tubes into one tube
7. Add 25 ul OB Protease solution to the tube
8. Vortex thoroughly the tube to promote the washing of the cells
9. Draw off 10 ul of cell solution plus 10 ul of iodine
10. Pipette 3, 2ul drops of he dyed cell solution on a concavity slide
11. count the cysts (dark round objects) in each drop for each drop

Lysis:

1. Add 220 ul of BL buffer to the tube of washed cells. (releases DNA under denaturing conditions, so proteins and enzymes are inactivated)
2. Vortex the tube thoroughly to make sure the contents were well mixed
3. incubate the tube at 70 C for 10 minutes in heat block
4. Vortex briefly after incubation

Binding:

1. Add 220 ul of 100% ethanol to the tube
2. Vortex to mix thoroughly
3. Insert a HiBind DNA Mini Column into a 2 mL Collection tube
4. Transfer the sample with ethanol to the HiBind DNA Mini Column
5. Centrifuge at maximum speed of 13,000 x g for 1 minute.
6. Discard the liquid from the collection tube
7. reuse the collection tube, clean the ethanol solution from the tube

Wash and Dry:

1. Add 500 ul HBC Buffer to the column (buffer is a diluted chatropic buffer that removes the impurities from the column while leaving the DNA bound to the silica)
2. Centrifuge at maximum speed, 13,000 x g,  for 1 minute.
3. After the HBC Buffer wash, discard the filtrate and collection tube
4. Insert the HiBind DNA Mini Column into a new, clean 2 mL Collection Tube
5. Add 700 ul DNA Wash Buffer (Buffer removes the salts from the DNA sample)
6. Centrifuge at maximum speed for 30 seconds
7. Discard the filtrate and reuse the collection tube
8. **Repeat the steps for a second DNA Wash Buffer wash step to ensure the DNA is clean
9. Centrifuge the empty HiBind DNA Mini Column at 13,000 x g for 2 minutes to dry the column and remove any left other ethanol

Elute:

1. Transfer the Column into a 1.5 mL microcentrifuge tube and lable
2. Add 100 ul of the heated Elution Buffer to the column
3. Let the tube sit at room temperature for 2 minutes
4. Centrifuge one last time at 13,000 x g for 1 minute. Your DNA is now in the microfuge tube in liquid form
5. Store eluted DNA at -20 degrees C

RESULTS:

observed cells at 40X: There are lots of round objects, hard to count because so many particles, the darker rounder objects are the cysts, cysts contain multiple ciliates sometimes.

Cell count drop #1: 46 cysts

Cell count drop #2: 51 cysts

cell count drop #3: 48 cysts

2ul= 48 cells

1ul=24 cells

about 125ul=3,000 cysts in the solution after cleansing

The HiBind DNA Mini Column had a ring of dark substance (dirt) at the filter which may prevent from good filtration. 

CONCLUSION: We don’t know if this protocol is accurate for soil ciliate as for the dirt residue tend to clog the filter. We know that there were cysts in our solution since we calculated about 24,000 cells per mL. We will see if there is any DNA present in our solution next class when we perform PCR on the solution. We got through the whole protocol and stored our DNA at -20C. Next class we will check for the presence of DNA so we can then perform PCR. If this protocol didn’t work we will have to try another that in more appropriate for soil.