Purpose: 

The purpose of the lab was to extract cells in the pellet we obtained from the previous lab.

Procedure:

Before starting:

• Set heat block to 70° C
• Heat Elution Buffer to 70° C in the heat block
• Chill PBS to 4° C

Cell counts:

• Spin sample for 5 min at 3000 x g
• Remove supernatant using a p1000 and discard
• Add 1ml of PBS, washing the cells
• Spin for 5 min at 3000 x g
• Remove supernatant using p100 and discard
• Put 100 microliters PBS in each tube to resuspend cells, then combine the two samples into one tube
• Put 20 microliters of Iodine and 20 microliters of cells in a new tube and vortex
• Take 3, 2 microliter drops and examine them under the microscope. Record cell counts
• Combine 20 microliters of Iodine and 20 microliters of cells in a new tube and vortex briefly

Prepare the cell suspension:

1. Wash the cells with cold PBS
2. Resuspend cells in 200 μl of fresh PBS
3. Add 25 μl OB Protease Solution. Vortex to mix thoroughly.

Lysis:

5. Add 220 μl BL Buffer
6. Incubate at 70°C for 10 minutes in the heat block. Briefly vortex the tube once during incubation.

Binding:

6. Add 220 μl 100% ethanol. Vortex to mix thoroughly
7. Insert a HiBind. DNA Mini Column into a 2 mL Collection Tube
8. Transfer the entire sample from Step 5 to the HiBind. DNA Mini Column including any precipitates that may have formed
9. Centrifuge at maximum speed for 1 minute
10. Discard the filtrate and reuse the collection tube

Wash and Dry:

11. Add 500 μl HBC Buffer to the column
12. Centrifuge at maximum speed for 30 seconds
13. After the HBC Buffer wash, discard the filtrate and collection tube.
14. Insert the HiBind DNA Mini Column into a new 2 mL Collection Tube.
15. Add 700 μl DNA Wash Buffer.
16. Centrifuge at maximum speed for 30 seconds.
17. Discard the filtrate and reuse the collection tube.
18. Repeat the steps for a second DNA Wash Buffer wash step
19. Centrifuge the empty HiBind DNA Mini Column at maximum speed for 2 minutes to dry the column.

Elute:

20. Transfer the HiBind DNA Mini Column into a nuclease-free 1.5 mL microcentrifuge tube. Make sure to label your tube with your name and date
21. Add 100μl Elution Buffer heated to 70°C
22. Let sit at room temperature for 2 minutes.
23. Centrifuge at maximum speed for 1 minute
24. Store eluted DNA at -20°C.

Data:

Under the microscope, I saw a yellow solution with a bunch of red dots and I ended up diluting it by a factor of 1:100 because it was too much to count and the drop I had was about 22 cells so that means there are a total of 2,200 cells in the 200 microliter suspension.

The microfuge tube was labeled SAM 2/15 on the top and group 2.7 DNA on the side.

The final sample was clear in color.

Conclusion:

In conclusion, the experiment was successful and the next step is to extract the DNA.