Week of 10/28/24-11/1/24

Intro

This week we started with RNA extraction, then had to do another round of desiccation to, hopefully, get better numbers. We also discussed this week the make of our rehydration buffer, as the recipe has changed slightly and after that change we have seen a decline in our results.

Methods

Pre-desiccation cell packing 

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

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

Results & Discussion

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.

Conclusion

With new cells in the process of desiccating, next week we will start the cycle over again. There have been talks of lowering the amount of beadbeating we do in order to increase our overall yield and other ways we can hopefully keep our cells from being lost during the proceure.

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