Week of 10/14/24-10/18/24

Intro

This week involved another round of RNA isolation, followed by purification and then an RNA gel and fluorometer check to ensure quality of the RNA before proceeding. 

Methods

Before RNA isolation, cells were rehydrated with 650uL of rehydration buffer per 100uL spot, then allowed to sit on the rocker for 15minutes at 250rpm to fully rehydrate. Kapton squares were then scraped to ensure removal of the cells, then pipetted into 1.5mL eppendorf tubes.

RNA isolation procedure:

1. Resuspend fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bashingbead lysis tube
2. Secure the tube on a bead beater fitted with a 2ml tube hold assembly and process. 1 min on/2 min ice repeated 5 times
3. centrifuge the tube for one minute to pellet debris
4. transfer up to 400μl of the cleared supernatant into a Zymo-Spin IICG column in a collection tube and centrifuge for one minute. Save the flow through
5. Add an equal volume of ethanol (95-100%) and mix well
6. transfer the mixture into a Zymo-spin IICR column in a collection tube in centrifuge for one minute. Discard the flow through
7. add 400μl RNA wash buffer to the column and centrifuge for one minute, then discard the flow through
8. prepare the DNase 1 reaction mix (75μl DNase buffer and 5μl DNase per tube) and add 80μl directly into column matrix and incubate at room temperature (20-30°C) for 15 minutes, then proceed with purification
9. add 400μl RNA prep buffer into the column and centrifuge for one minute. Discard the flow through
10. add 700μl RNA wash buffer into the column and centrifuge for one minute. Discard the flow through
11. add 400μl RNA wash buffer into the column and centrifuge for one minute. Discard the flow through
12. add 400μl RNA wash buffer into the column and centrifuge for two minutes. Discard the flow through
13. transfer the column into a nucleus free tube, and add 50μl DNase/ RNase free water directly to column matrix and centrifuge

RNA clean up procedure:

1. Add 2 volumes RNA binding buffer to each sample and mix
2. Add an equal volume of ethanol (95-100%) and mix
3. Transfer the mixture into a Zymo-spin IICR column in a collection tube in centrifuge for one minute. Discard the flow through
4. add 400μl RNA prep buffer into the column and centrifuge for one minute. Discard the flow through
5. add 700μl RNA wash buffer into the column and centrifuge for one minute. Discard the flow through
6. add 400μl RNA wash buffer into the column and centrifuge for one minute. Discard the flow through
7.  add 400μl RNA wash buffer into the column and centrifuge for two minutes. Discard the flow through
8. transfer the column into a nucleus free tube, and add 50μl DNase/ RNase free water directly to column matrix and centrifuge

RNA gel was made with a 1% TAE solution that was allowed to cool slightly before adding 2uL of midori green for visibility of RNA.

Results & Discussion

Results for the samples were as follows before the cleanup:

ng/ul

a260/280

260/230

A

35.32.010.53

B

85.21.951.54

C

133.0

1.670.60

Results for the samples after the purification procedure were as follows:


Samples were much more pure after the purification procedure but did unfortunately sacrifice much of the yield.

Conclusion

With such low yield, we decided that we will have to start again with growing new samples and continuing with the extraction procedures.

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