RNA Isolation

    This week was a short week as i was out for a few days. Most of the time spent in lab was spent helping the new people learn various skills or equations they will be using in lab. i was able to redo the RNA isolation, but overall most of the time i had in lab was spent teaching. We covered things like making media, C1/V1 equations and the nanodrop as well as things like general lab layout and where to find various necessities. 

    We were also able to talk more about the issues with the organic contamination and after reviewing the procedure it was realized that while the RNA isolation was normally done on Deinococcus, E.coli did not have the same strength cell wall. While ten cycles of bead beating was necessary to break open the Deinococcus cells, the same amount of cycles was destroying the E.coli cells and causing the high levels of organic contamination we had seen.

    The UV exposure was performed at 70 mJ/cm^2 for two minutes, with the E.coli being grown that morning then normalized, exposed and diluted just like the week before. After the 30 minute recovery at 30°C we moved on to RNA isolation. For the most part the procedure was the same however with our A control/test pair we did one round of beat beating, with the B control/test pair we did two rounds and with the C control/test pair we did 5 rounds. The rest of the procedure was done the same as last week.

Starting OD values and normalized values

Sample

OD

Normalized OD

A

1.42

1.04

B

1.30

0.98

C

1.47

1.02

RNA isolation

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

1.     2. Secure the tube on a bead beater fitted with a 2ml tube hold assembly and process. 1 min on/2 min ice repeated 1, 2 and 5 times

1.     3. Centrifuge the tube for one minute to pellet debris

1.     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

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

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

1.     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

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

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

1.     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 one minute. 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



Tube

ng/μl

A260/A280

A260/A230

A control

105.1

2.10

1.14

A test

64.7

2.13

1.38

B control

52.5

2.18

1.31

B test

63.8

2.13

1.59

C control

106.4

2.09

1.28

C test

21.5

2.17

1.25

There was still organic contamination, however it was much less so the beat beating was one of the issues. Next week we will perform an Ethanol precipitate to see if we can get rid of some of the organic contaminates and hopefully use the samples for the LexA qPCR. 

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