RNA Isolation


We started Monday by doing a RNA isolation. Before we could begin the actual RNA Isolation procedures, we needed to first dilute our samples (close to 1.00 ng/μl) and expose the test cells to Hydrogen Peroxide.

Post Dilution Values-
T1 1.04 A600
T2 1.04 A600
T3 1.07 A600
C1 1.04 A600
C2 1.04 A600
C3 1.04 A600

After Dilution, we exposed T1, T2 and T3 to Hydrogen Peroxide. We had 200 μl of sample, and added 200 μl of H2O2 (200 mM) for the 1:1 ratio of sample to H2O2 needed. We also added 200 μl of media to C1, C2 and C3 in order to keep the volume consistent. After adding either H2O2 or Media to all of the samles, we incubated at 30°C for 30 minutes.

After thoroughly cleaning the inside of the hood, and all the equipment needed, we moved on to the actual RNA Isolation Procedures. 


RNA Isolation Procedures-

We start by centrifuging and resuspending the cell pellet in 800 μl of RNA Lysis buffer and transfer the mixture into a glass bead beating tube.

We then put the tubes into a bead beater for 1 minute, than onto ice for 2 minutes, and repeat the process 10 times. 

After centrifuging for a minute we transfer 350 μl of the supernatant from the beat beating tube into a spin column and centrifuge again.

We then add a 1:1 ratio of 95-100% Ethanol to the flow-through and transfer to another spin column, and centrifuge again, this time discarding the flow-through. 

 Next, add 400 μl of RNA wash buffer to the column, centrifuge and discard the flow-through. 

Add 75 μl of DNA digestion buffer and 5 μl DNase 1 to the column and incubate at 30°C for 15 minutes. 

We then add 400 μl of RNA Prep buffer, centrifuge and discard flow-through. 

After we add 700 μl of RNA wash buffer and centrifuge, and discard flow-through.

Next we add 400 μl of RNA wash buffer and centrifuge, and repeat that step again. 

Finally we transfer the column to a nuclease free tube, add 50 μl DNase/RNase free water and centrifuge. 



We then nanodropped our six samples-

 

 ng/μl

 A260/280

 A260/230

 T1

 55.6

 1.68

 .65

 T2

 328

 1.6

 .50

 T3

 15

 2.19

 1.10

 C1

 14.3

 2.13

 1.64

 C2

 39.7

 1.72

 .47

 C3

 123

 1.66

 .53

Ideally, we want our ng/μl to be over 50, our A260/280 over 2.0 and our A260/230 over 2.0



Tuesday we decided to delegate specific things to different team members depending on the days they were in lab. While these will change in the future, here are our current jobs-

RNA isolation- Deja
Growing Cells- Kholoud
Loading Plates- Alex
Analysis- Malica
cDNA Synthesis- Alex
Quality Control- Malica.

While we will all do various tasks and occasionally do different jobs, these are our main tasks currently. 


Wednesday we did a graham stain on our culture as well as make some media for the bio 175 lab. 




Friday we once again did a RNA isolation. 

We started by doing a mass dilution of our samples instead of diluting each tube.


After dividing up our diluted samples into six tubes, we exposed the test tubes to H2O2, added media to the controls and incubated. 

We did the same protocol as normal, with the difference of only removing 300 μl from the bead beating tube instead of 350 μl. 

The T1 tube was knocked over shortly before adding the ethanol, losing about 100 μl, and as the ethanol was 1:1 that volume was different than that added to the rest of the tubes. However the rest of the steps were identical for all of the tubes. 



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