Bio443
Expt 3: X-Chromosome Inactivation Experiment
Collection of cells
1. Collect 15 ml of cell suspension for each of the 4 lymphoblast cell lines.
2. Centrifuge for 2 min at 1000 g.
3. Discard the media and resuspend the pellet in 3 ml of PBS.
4. Place 1.5 ml of cell suspension into each of 2 microfuge tubes.
5. Centrifuge for 1 min at 12,000 g.
6. Discard the PBS and invert over a papertowel to remove excess liquid.
7. One pellet is used for RNA isolation.
8. The other pellet is used to DNA isolation
RNAqueous-4PCR kit from Ambion
RNA Isolation from Eukaryotes
1. Discard the supernatant and add 500 ul of lysis/binding solution.
2. Resuspend the cell pellet by pipetting up and down and transfer the cell suspension to a new RNase
free microfuge tube. Incubate on ice for 5 minutes.
3. Centrifuge the microfuge tube at 14K rpm for 2 minutes to pellet cell debris.
4. Transfer the cell lysate to a new microfuge tube.
5. Add an equal volume (500 ul) of the 64% Ethanol and mix by carefully pipetting.
6. Apply 700 ul of the lysate/ethanol mixture to a filter cartridge/collection tube.
7. Centrifuge for 1 minute at 14K rpm.
8. Discard the flow-through and repeat steps 8-10.
9. Apply 700 ul of Wash solution #1 to the filter cartridge and centrifuge for 1 minute at 14K rpm.
10. Discard the flow-through.
11. Apply 500 ul of Wash solution #2/3 and centrifuge as above.
12. Repeat Wash solution 2/3 step with 500 ul.
13. Discard the flow through and centrifuge once more to remove any residual wash solution.
14. Put the filter cartridge in a new collection tube.
15. Place 60 ul of preheated (70-80C) elution solution onto the center of the filter.
16. Centrifuge for 1 minute.
17. Using the same collection tube, add another 40 ul of preheated elution solution to the filter and
centrifuge for 1 minute.
18. Place the collection tube on ice.
19. Determine the concentration of your RNA samples on the Nano-drop.
DNA Isolation from Eukaryotes
1. Add 600 ul of Nuclei Lysis Solution and completely resuspend by pipeting.
2. Place the microfuge tube in the -80C freezer for 10 minutes.
3. Thaw the tube and vortex.
4. Add 3 ul of RNase Solution to the tube, mix by inverting and incubate at 37C for 15 minutes.
5. Let the tube cool to room temperature and ass 200 ul of Protein Precipitation solution .
6. Vortex at high spped for 20 seconds and incubate on ice for 5 minutes.
7. Centrifugre for 4 minutes at max speed.
8. Carefully remove the supernatant containing the DNA and transfer it to a clean tube containing 600
ul of room temperature isopropanol.
9. Gently mix and centrifuge at max speed for 1 minute.
10. Carefully remove the supernatant and add 600 ul of 70% ethanol.
11. Centrifuge for 1 min at max speed.
12. Carefully remove the supernatant and invert over a paper towel to air dry.
13. Add 100 ul of DNA Rehydration Solution and incubate at 65C for 1 hour.
14. Store the DNA at 65C.
First-Strand cDNA Synthesis
1. Calculate the volume of your RNA necessary to have 5.0 ug.
2. Each group will set up one cDNA reaction, using the following reaction set-up in a 0.2 ml
microfuge tube.
10.0 ul
3.0 ul
1.0 ul
____ ul
____ ul
20.0 ul
2X cDNA synthesis master mix
Oligo (dT) primer
AffinityScript RT/RNase Block enzyme mixture
RNA
RNase-free H2O (bring up to 20 ul)
Total Volume
3. Incubate at room temperature for 5 minutes and then for 42C for 30 minutes.
4. Place the reaction mix at 95 for 10 minutes to degrade the RT.
Polymerase Chain Reaction (PCR)
1. Each group will set up 8 reactions
12.5 ul
0.5 ul
0.5 ul
1.0 ul
10.5 ul
25.0 ul
2. Run thermocycler.
2X PCR Master Mix
Forward primer
Reverse primer
DNA template OR 2.0 ul cDNA template
sterile H2O
Total Volume