Purpose: To extract DNA from our ciliates by Ludox extraction

Materials:

• Tabletop microcentrifuge capable of 13,000 x g
• Nuclease-free 1.5 mL microcentrifuge tubes
• Heat block capable of 70C
• Vortexer
• 100% ethanol
• 100% isopropanol
• PBS (or other wash buffer)

Before Starting

• Set heat block to 70C
• Prepare DNA wash buffer and HBC buffer
• Heat elution buffer to 70C in heat block
• Chill PBS to 4C (fridge temperature)

Prepare Cell Suspension

1. Wash the cells with cold PBS. (This means add 200ul PBS, resuspend the cells using the vortex or flicking, and then spin and remove the supernatant carefully without disturbing the pellet)
2. Resuspend cells in 200 ul of fresh PBS
3. Add 25 ul OB Protease solution. Vortex to mix thoroughly

Lysis

1. Add 220 ul BL Buffer. (BL Buffer has an important role in nucleic acid extraction by destabilizing molecular bonds which leads to destabilization of proteins)
2. Incubate at 70C for 10 minutes in the heat block. Briefly vortex the tube once during incubation

Binding

1. Add 200 ul 100% ethanol. Vortex to mix thoroughly.
2. Insert a HiBind. DNA Mini Column into a 2 mL collection tube
3. Transfer the entire sample from step 2 of the ‘Lysis’ to the HiBind. DNA Mini COlumn including any precipitates that may have formed
4. Centrifuge at maximum speed (>10,000 x g) for 1 minute
5. Discard the filtrate (liquid that went into collection tube) and reuse the collection tube. Some proteins and polysaccharides will flow through, but some will remain on the column

Wash and Dry

1. Add 500 ul HBC Buffer to the column. This will remove the impurities from the column while leaving DNA bound to silica. (Note: HBC buffer diluted with 100% isopropanol)
2. Centrifuge at maximum speed for 30 seconds
3. After the HBC Buffer wash, discard the filtrate and collection tube
4. Insert the HiBind DNA Mini Column into a new 2 mL collection tube
5. Add 700 ul DNA wash buffer (Note: DNA wash buffer is diluted with 100% ethanol; this removes salts from DNA sample)
6. Centrifuge at maximum speed for 30 seconds
7. Discard the filtrate and reuse the collection tube
8. Repeat steps for a second DNA wash buffer wash step. (this removes last bit of salt impurities)
9. Centrifuge the empty HiBind DNA Mini Column at maximum speed for 2 minutes to dry the column

Elute

1. Transfer the HiBind DNA Mini Column into a nuclease-free 1.5 mL microcentrifuge tube
2. Add 100 ul elution buffer heated to 70C. (for DINA extraction, 10 mM Tris at pH 8-9 is typically used which makes DNA more stable which will dissolve faster)
3. Let sit at room temperature for 2 minutes
4. Centrifuge at maximum speed for 1 minute. DNA is now in the liquid flow through and column may be discarded
5. Store eluted DNA at -20C.

Results/Conclusion:

Our protocol ended up going very smoothly. When we finished the first cell suspension step, we found that our solution ended up being fairly pure with only a little soil and a little clay found in our solution. We encountered a problem in the last step of our protocol. The DNA was, unforunately, accidentally thrown out into the trashcan instead of being kept in the tube. Instead of repeating our Ludox centrifugation and DNA extraction protocol, we repeated the last step with the hopes that there was still some DNA left on top of the filter in the HiBind DNA Mini Column. Next lab, we are hoping even though it may not be as much, there is DNA in the solution we collected.