Protocol 17 - Isolating RNA, DNA and Protein from Drosophila in Sequence
Supplies needed:
- 1.5 ml microcentrifuge tubes
- Trizol
- chloroform
- isopropyl alcohol
- 80% ethanol
- dd H2O
- 0.1% sodium citrate in 10% ethanol
- 100 % ethanol
- dialysis tubing
Equipment needed:
- heat block or hot water bath at 30°C
- microcentrifuge
- P-10, 100, 1000 micropipettors
Protocol A: RNA Isolation
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1. Freeze 50 Drosophila larvae or flies in a microcentrifuge Tube P (pellet) on dry
ice or wet ice to slow down the fly movement.
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2. Grind the 50 larvae or flies in 500 μl of Trizol with a grinding rod (pellet
pestle). Rinse the rod off with an additional 500 μl of Trizol.
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3. Incubate at 30°C for five minutes in heat block or water bath.
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4. Centrifuge at 12,000 rpm for 5 minutes. You will have a pellet of fly debris and
organic molecules dissolved into the Trizol.
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5. Transfer the 1 ml of the pinkish Trizol with organic molecules into a new Tube
S (supernatant) without disturbing the pellet at the bottom. [NOTE: Save Tube P
with the pellet to return to it in steps below.]
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6. Add 200 μl of chloroform to Tube S and shake vigorously for 15 seconds.
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7. Incubate at 30°C for thee minutes in heat block or water bath.
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8. Centrifuge Tube S at 12,000 rpm for 5 minutes. At the end of the
centrifugation, you should observe: 1) a pink/reddish organic phase (OP) at the
bottom 2) a small white interphase (IP) with amphipathic proteins soluble in both
water and lipid 3) and upper clear aqueous phase (AP).
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9. Carefully separate the three phases the upper clear aqueous phase into three
new tubes labeled S-AP (aqueous phase), S-IP (interphase), and S-OP (organic
phase). [Note: Save the S-IP and S-OP for later steps in the isolation.]
-----------Back to original Tube P with the Pellet--------------□
10. Add 200 μl of Trizol to the pellet in Tube P and once again grind with a
grinding rod (pellet pestle). Then add another 800 μl of Trizol.
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11. Add 200 μl of chloroform to Tube P and shake vigorously for 15 seconds.
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15. Incubate at 30°C for thee minutes in heat block or water bath.
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16. Centrifuge Tube S at 12,000 rpm for 5 minutes. At the end of the
centrifugation, you should observe: 1) a pink/reddish organic phase (OP) at the
bottom 2) a small white interphase (IP) with amphipathic proteins soluble in both
water and lipid 3) and upper clear aqueous phase (AP).
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17. Carefully separate the three phases the upper clear aqueous phase into three
new tubes labeled P-AP (aqueous phase), P-IP (interphase), and P-OP (organic
phase). [Note: Save the P-IP and P-OP for later steps in the isolation.]
----Now return to the aqueous phase tubes Tube S-AP and Tube P-AP to isolate RNA----□
18. Add 500 μl of isopropyl alcohol to Tubes #S-AP and #P-AP. Recall that these
two tubes contain the aqueous phase the harbors the RNA we seek to isolate.
Gently invert the tube to mix. This alcohol will cause precipitation of the RNA.
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19. Incubate at 30°C for 10 minutes in heat block or water bath.
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20. Centrifuge at 12,000 X for 10 minutes.
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21. Decant off the supernatant being careful not to disturb the RNA pellet.
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22. Add 100 μl of 80% ethanol to rinse off the RNA pellet and then spin in the
microcentrifuge at 14,000 RPM to once again pellet the RNA at the bottom of the
tubes.
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23. Once again, add 100 μl of 80% ethanol to rinse off the RNA pellet and then
spin in the microcentrifuge at 14,000 RPM to once again pellet the RNA at the
bottom of the tubes. Let the alcohol dry out.
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24. Add 50 μl of dd H2O and resuspend the RNA pellet in sterile dd H2O.
Protocol B: DNA Isolation (using tubes S-OP and P-OP from Protocol A above)
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1. Your DNA and protein is in the organic phase of the from Protocol A above.
Add 300 μl of 100% ethanol and mix in the alcohol by gently inverting the tube
several times.
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2. Incubate at 30°C for 3 minutes in heat block or water bath.
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3. Centrifuge at 12,000 X for 15 minutes. Your pellet will contain your DNA
while the supernatant will retain the proteins. Remove the supernatant and transfer
it to Tube Pro (Protein) and save it for the protein isolation Protocol C below.
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4. Wash the pellet with 1 ml of 0.1% sodium citrate in 10% ethanol for 30
minutes at 30°C.
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5. Centrifuge at 2,000 X for 5 minutes and observe the pelleted DNA.
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6. Once again, wash the pellet with 1 ml of 0.1% sodium citrate in 10% ethanol
for 30 minutes at 30°C for 30 minutes.
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7. Centrifuge at 2,000 X for 5 minutes and observe the pelleted DNA.
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8. Decant off the supernatant being careful not to disturb the DNA pellet.
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9. Add 50 μl of dd H2O and resuspend the DNA pellet in the water.
Protocol C: Protein Isolation (using the Tube Pro from Protocol B above)
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1. Dialyze the supernatant in Tube Pro against 3 changes of 0.1% SDS
solution at 2°C to 8°C.
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2. Centrifuge the dialyzed supernatant at 10,000 X for 10 minutes.
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3. Reserve the clear supernatant for Western Blotting.