Deinococcus sonorensis and qPCR

 Introduction

   This week, most of our time was dedicated to the sonorensis project, as well as a discussion into the basics of qPCR. Most of the time spent with sonorensis was discussing what further tests need to be completed and/or done again; the time spent on the AMC project was spent doing another run of RNA isolation.

Methods

Exposure

  1. Take 100μl of sample and expose to 200mJ/M^2 of UV radiation for treatment period. (5min &10 min in this case) 

  2. After exposure take the 100μl and add it to 400μl of TGY

  3. Incubate at 30°C for 30 minutes before moving on to RNA isolation 


RNA isolation

  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 10 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

Sonorensis Characterization tests:

  • sugar fermentation- tests for production of fermentation products from sugars such as lactose and sucrose (typically acids, indicated by color change via pH indicator)
  • MRVP- two tests in one, MR&VP. Inoculate 3 separate vials, one positive, one negative, one w/ sonorensis; with methyl red, color of pink/red is a positive result; add bar A for VP portion and a positive result is red color while a negative is any other color/change
  • starch- make starch plate, inoculate w/ +, -, and sono. Add iodine, black=negative, halo zone= + for amylase
  • citrate- make citrate plate, add +, -, sono, positive result= color change from green to blue. Tests for citrate permease
  • oxidase- 4 way streak sono, add oxidase to each section, observe for color change (cytochrom-c oxidase, red --> purple)
  • catalase- add 2 drops 3% hydrogen peroxide to largest colony. If positive, bubbles (oxygen production)
  • SIM- sulfur, indole, motility; indole looks for tryptophase (+=red); agar will turn black if positive for sulfur; sample spreads through agar if motile, makes hazy zone
  • sporulation- starve bacteria to see if endospore production occurs and/or has tougher cell wall
  • urea- if positive for urease, will turn pink (alkaline)
  • casein- protease, looking for clear halo

Results

No results due to issues with the e. coli sample used for the round of RNA isolation so it could not be completed.

Discussion

As we continue to work at RNA isolation and towards qPCR for the AMC project, it is important to know about the basics of qPCR and to understand how it works and what information it gives. In very basic terms, qPCR is used to show gene expression over time. DNA is isolated , then denatured and copied. Dyes intercolate and give off a phosphorescent signal that builds up over time and shows up within the graph earlier or later based upon how much the gene is expressed. There are different variations of qPCR, such as one-step, which has less variation and thus lower risk of contamination, with high throughput; essentially you get more data over less time. Two-step has more priming options, more reactions per sample, and gives a wider scope of the genes.

Conclusion

The plan moving forward is to continue with RNA isolation to continue seeing qPCR and results within. Many tests need to be done for the characterization of sonorensis so it is very likely that I'll be helping out with those as well, and hopefully we should be able to start with poster planning.

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