Isolation and Desiccation

 We started this week off with an RNA Isolation, then prepared for and performed a pre-desiccation procedure later on. We did the usual RNA isolation procedures on samples A B and C. Last week the samples were prepared then moved to the Egg for true desiccation after 24 hours of drying. They then underwent true desiccation for 6 days before rehydration and RNA isolation. The wells were rehydrated using 650ul of the rehydration buffer, then put on he shaker for 15 minutes. After that we used inoculation loops to scrape the cells before centrifuging them to pellet for the RNA isolation procedure.


RNA:

1. Resuspend a fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bead bashing lysis tube

2. Secure the tube on a bead beater with a 2ml tube hold assembly and process. Repeat 5 cycles of 1 minute on and 2 minutes on ice

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. Retain the flow through

5. Add an equal volume of ethanol (95-100%) and mix thoroughly

6. Transfer the mixture into a Zymo-spin IICR column in a collection tube and centrifuge for one minute. Discard the flow through

7. Add 400μl RNA wash buffer to the column, centrifuge for one minute, and discard the flow through

8. Prepare the DNase 1 reaction mix (75μl DNase buffer and 5μl DNase per tube), add 80μl directly to the column matrix, and incubate at room temperature (20-30°C) for 15 minutes before continuing purification

9. Add 400μl RNA prep buffer to the column and centrifuge for one minute. Discard the flow through

10. Add 700μl RNA wash buffer to the column and centrifuge for one minute. Discard the flow through

11. Add 400μl RNA wash buffer to the column and centrifuge for one minute. Discard the flow through

12. Add 400μl RNA wash buffer to the column and centrifuge for two minutes. Discard the flow through

13. Transfer the column to a nuclease-free tube, add 50μl DNase/RNase-free water directly to the column matrix, and centrifuge



ng/ul

a260/280

260/230

A

20.52.071.20

B

32.31.891.03

C

37.5

1.941.56


While the 260/230 values are good, the 260/280 values are to low meaning we have to many organic contaminates, most likely either from the rehydration solution or the salt from the various washes and buffers in the kit. This usual wouldn't be an issue as we would be able to perform a clean up, but the ng/ul were already to low. Its been decided that volumes under 65ul wouldn't be acceptable as there is minimum amounts needed for the various quality checks and sequencing procedures, and less than 65 wouldn't be enough.

We also inoculated a set of flasks that were able to grow for 48 hours before moving on to pre-desiccation for another set. After two days dehydrating they were placed in the Egg for true desiccation and left there for the weekend. 

1. Normalize 3ml of culture to an OD between 0.95-1.00

2. Spin down 1ml of media, remove supernatant and resuspend pellet in nuclease free water

3. Resuspend pellet, then centrifuge and remove supernatant

4. Add the second ml of culture, repeat washing steps

5. Add the third ml of culture, repeat washing steps

6. Plate 100ul dots into 1 inch kapton squares in a 6 well plate in triplicate


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