First week: Testing E.coli UV Tolerance and RNA Isolation

     This week was prepping for the first LexA E.coli qPCR, meaning we first had to make sure that the UV limits were correct then perform an RNA isolation. The beginning of the week was spend busy with biotech bootcamp for the first few days, but after that i got started with the UV exposure to ensure that the levels that we expected to see significant impact wouldn't kill them. For this i did a simple experiment by exposing them at three different levels of UV radiance (50, 60 and 70 mJ/cm^2) for two different times (1 and 2 minutes. All of the samples were exposed then plated alongside a control, and grown till the next day. All of the plates had growth so we were able to use the highest levels and times at 70 mJ/cm^2 for two minutes during our RNA isolation. 

    The next day I did the RNA isolation, diluting the samples grown that morning (due to the doubling time of E.coli) to an OD of 1 +/- .05, then exposing 100μl and after letting them recover in 900μl of LB broth getting a 1 in 10 dilution. After a 30 minute recovery at 30°C I was able to move on with the standard RNA isolation procedure. 


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 10 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


        The RNA isolation was performed as normal, however at the end there was a high level of organic contamination. In the end it was ruled that an alcohol precipitate would not be a good choice to do on this sample and they were instead thrown out. At this point the reason for the contamination is unknown, but I have made sure to clean out the biological hood thoroughly with the correct supplies as well as checked all of the dates of the solutions to ensure that those weren't the cause. 


Tube

ng/μl

A260/A280

A260/A230

A control

76.2

1.83

0.54

A test

64.8

1.81

0.40

B control

72.6

1.86

0.46

B test

96.3

1.80

0.53

C control

53.4

1.78

0.70

C test

43.2

1.85

0.61

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