BISC220/S11: Mod 3 Lab 9

Apoptosis

Apoptosis

Genomic DNA Isolation Protocol

Cells were treated with either 500 uM etoposide (VP-16) or as a control DMSO alone over a time course of 8 hours. Samples were taken at 2 hour intervals (0, 2, 4, 6 and 8). At each interval 1x10⁶ cells were pelleted in a microcentrifuge.

The following two steps have been done for you.

1. Wash the cells twice with 1X PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 2 mM KH₂PO₄, pH 7.2).
2. Washed cells were resuspended in DNA extraction buffer (50 mM Tris, pH 8.0, 20 mM Na₂EDTA, 10 mM NaCl, 1% [w/v] SDS, and 20 μg/ml RNase A) and incubated at 37°C for 1 hour to lyse the cells and digest cellular RNA.
3. Then Proteinase K was added to a final concentration of 100 ug/ml and incubated at 65°C for 1 hour to digest cellular proteins.
   

To do in lab in the HOOD:

1. Add 250 ul of Trizol reagent to the cell extract. Mix by inversion a few times and let incubate at room temperature for 5 minutes in the hood.
2. Add 100 ul chloroform to each sample. Mix by shaking for 10 seconds and incubate for 2 minutes in the hood.
3. Spin at top speed in a microfuge at 4°C for 15 minutes.
4. Move the clear aqueous phase to a new microfuge tube. DO NOT pipette any of the pink phenol. It is OK to leave a little of the aqueous phase behind to prevent getting any of the phenol.
5. Dispose of the phenol in the marked container in the hood. Put the empty microfuge tube in the marked bio-hazard bag.
6. Estimate how much liquid is in your microfuge tube.
7. Add 2 volumes of ICE COLD ethanol to your sample.
8. Mix by shaking for a few seconds. Look at your sample - you should see small bubbles throughout your sample. That is the DNA precipitating.
9. Put your samples at -80°C for 20 minutes. This helps to further precipitate your DNA.
10. Spin your samples in a microfuge at the highest speed for 20 minutes to pellet the DNA.
11. Remove the supernatant and put that in a fresh microfuge tube to save just in case.
12. Look on the sides of your tubes - can you see a pellet?
13. Wash the pellet by gently adding 700 ul of 70% ethanol to the side of the tube opposite your pellet.
14. Spin for 3 minutes at top speed to be sure your pellet was not dislodged.
15. Remove the ethanol and discard - being careful not to disrupt the pellet.
16. Leave the tube open and upside-down on a paper towel on your bench for about 5 minutes to allow the ethanol to evaporate.
17. Add 40 ul of TE to your pellet.
18. Pipette up and down gently a few times to resuspend the pellet.

Agarose Gel Electrophoresis Protocol

Prepare a sample for electrophoresis by mixing 10 μL of your genomic DNA with 2 μL of loading dye on a 1.0% agarose gel with SybrSafe™ stain. Run the gel at 100V for 45 minutes. The gel will be photographed using UV light and the photo posted to Sakai.

Template for loading the gel
Lane 1: 1 kB ladder
Lane 2: Control 0 hours
Lane 3: Experimental 0 hours
Lane 4: Control 2 hours
Lane 5: Experimental 2 hours
Lane 6: Control 4 hours
Lane 7: Experimental 4 hours
Lane 8: Control 6 hours
Lane 9: Experimental 6 hours
Lane 10: Control 8 hours
Lane 11: Experimental 8 hours
Lane 12: 100 bp ladder

What size is genomic DNA? What does it look like on the gel? What happens to the genomic DNA over time?

Lab 8: Cell Culture
Lab 10: Apoptosis - Protein 1
Lab 11: Apoptosis - Protein 2
Lab 12: Imaging Presentations
Media Recipes