BIOLOGY 40-Protocol #2-Revised 6/18/07
Summer, 2007
Name:___________________________________
Genotype:________________________________
Protocol 2 - Isolating RNA, DNA and Protein from Drosophila in Sequence (Trizol Protocol)
Supplies needed:
Equipment needed:
- 1.5 ml microcentrifuge tubes
- heat block or hot water bath at 30°C
- Trizol
- microcentrifuge
- Chloroform
- P-10, 100, 1000 micropipettors
- Isopropyl alcohol
- dialysis tubing
- 80% ethanol
- dd H2O
- 0.1% sodium citrate in 10% ethanol
- 100 % ethanol
Protocol A: RNA Isolation
1. Freeze 8-10 Drosophila flies in the freezer (-20oC) or on dry or wet ice to slow down the fly
movement. Can prepare DNA from up to a maximum of 50 flies.
2. Grind the 8-10 flies in a microcentrifuge Tube P (pellet) with 500 μl of Trizol with a grinding
rod (pellet pestle). Rinse the rod off with an additional 500 μl of Trizol.
3. Incubate Tube P at 30°C for five minutes in heat block or water bath.
4. Centrifuge Tube P at 12,000 rpm for 5 minutes. You will have a pellet of fly debris and
organic molecules dissolved into the Trizol.
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.]
6. Add 200 μl of chloroform to Tube S and shake vigorously for 15 seconds.
7. Incubate at 30°C for three minutes in heat block or water bath.
8. Centrifuge Tube S at 12,000 rpm for 5 minutes. At the end of the centrifugation, you should
observe the following three (3) layers or phases:
1) Upper clear aqueous phase (SAP)
2) Small white interphase (SIP) with amphipathic proteins soluble in both water and lipid
3) Bottom pink/reddish organic phase (SOP)
BIOLOGY 40-Protocol #2-Revised 6/18/07
Summer, 2007
9. Carefully separate the three phases from Tube S into three new tubes labeled SAP (aqueous
phase), SIP (interphase), and SOP (organic phase). [Note: Save the SIP and SOP 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.
Note: The purpose of this step is to extract additional DNA/RNA from the pellet.
11. Centrifuge tube P for 5 minutes at 12,000 rpm.
12. Remove the supernatant from tube P and put into a new tube (P-S).
12. Add 200 μl of chloroform to Tube P-S and shake vigorously for 15 seconds.
13. Incubate tube P-S at 30°C for three minutes in heat block or water bath.
14. Centrifuge Tube P-S at 12,000 rpm for 5 minutes. At the end of the centrifugation, you
should observe three phases once again:
1) Upper clear aqueous phase (PAP)
2) Small white interphase (PIP) with amphipathic proteins soluble in both water and lipid
3) Bottom pink/reddish organic phase (POP)
15. Carefully separate the three phases the upper clear aqueous phase into three new tubes
labeled PAP (aqueous phase), PIP (interphase), and POP (organic phase). [Note: Save the PIP
and POP for later steps in the isolation.]
Note: Be sure to keep the SAP, SIP, SOP and PAP, PIP, POP tubes separated.
Now return to the aqueous phase tubes Tube SAP and Tube PAP to isolate RNA
16. Add 500 μl of isopropyl alcohol to Tubes #SAP and #PAP. 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.
17. Incubate at 30°C for 10 minutes in heat block or water bath.
18. Centrifuge at 12,000 X for 10 minutes.
19. Decant off the supernatant being careful not to disturb the RNA pellet.
20. 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.
BIOLOGY 40-Protocol #2-Revised 6/18/07
Summer, 2007
21. 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.
22. Add 50 μl of dd H2O* and resuspend the RNA pellet in sterile dd H2O.
Protocol B: DNA Isolation (using tubes SOP and POP from Protocol A above)
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.
2. Incubate at 30°C for 3 minutes in heat block or water bath.
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.
4. Wash the pellet with 1 ml of 0.1% sodium citrate in 10% ethanol for 30 minutes at 30°C.
5. Centrifuge at 12,000 X for 5 minutes and observe the pelleted DNA.
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.
7. Centrifuge at 12,000 X for 5 minutes and observe the pelleted DNA.
8. Decant off the supernatant being careful not to disturb the DNA pellet.
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)
1. Dialyze the supernatant in Tube Pro against 3 changes of 0.1% SDS solution at 2°C to 8°C.
2. Centrifuge the dialyzed supernatant at 10,000 X for 10 minutes.
3. Reserve the clear supernatant for Western Blotting.
Solution Preparation
First make a 1% sodium citrate solution by adding 0.5 g sodium citrate in 45 ml ddH2O,
and dilute to 50 ml.
To make 0.1% Sodium Citrate in 10% ethanol mix:
1 ml of 1% sodium citrate ()
1 ml of ethanol
8 ml of sterile ddH2O