Week 5: More Plasmid Extractions

 Introduction: 

Because of the lack of success from our plasmid extractions, we will be conducting more plasmid extraction but this time experimenting with different variables and independent factors that could've altered our results. As before, some of our plasmid extraction came out pretty good in terms of the concentration rate but unfortunately, did not show up well or even at all on a gel. Therefore, we will be redoing everything but with different independent factors. 

On 3/02/23, my partners and I decided to experiment with 4 different tubes, each having different procedures and its own factors, and we will see the different types of results. We have: 

--> Tube A: follows every step except the lysozyme procedure and no buffer (just get straight into the Zymopure Plasmid Kit), we also need to take aliquots of each wash (1+2+2) and put it into the nano drop. 

--> Tube B: follows every step but this time adding the lysozyme and lysis procedure (lysozyme+lysis+incubate for 30 minutes) 

--> Tube C: follows every step but extracting 700 ul of excess yellow from step 4 in our Zymopure Plasmid Kit into a new tube and adding 300ul of binding buffer. 

--> Tube D: our control. It follows every single steps of the whole procedure with no adjustments. 

I got to be tube A for this experiment. 

Procedure: 

1. I followed the plasmid extraction procedure like normally, excluding out the multi-buffer and lysozyme part. 

    -->  1 mL of D. Aquaticus + centrifuge to remove excess supernatant. 

2. I added 250 ul of ZymoPure P1 (red solution) to the tube and resuspending it completely 

3. I added 250 ul of Zymopure P2 (blue solution) and then invert for 8-10 times + 3 minute incubation 

4. I then added 250 ul of ZymoPure P3 (yellow solution) and inverting it until everything is fully mixed and yellow. 

5. I centrifuge the tube for 5 minutes at max power

6. I then transferred exactly 600 ul of the supernatant from step 5 into a clean 1.5 mL microcentrifuge tube. 

7. I added 260 ul of the ZymoPure Binding Buffer to the cleared lystate from step 6 and mix thoroughly by vortexing for 15 seconds. 

8. I place a Zymo-Spin II-PX column in a collection tube 

9. Then, I transfer the entire mixture from step 7 into the Zymo-Spin II-PX column. 

    --> incubating the tube at room temperature for 1 minute and then centrifuging for 1 minute. I discard the flow. 

10. I added 800 ul of ZymoPure wash 1 to the Zymo-Spin II-PX column. 

    --> I centrifuged it for 1 minute 

    --> Before I discard the flow, I aliquot it. I got -2.3 ng/ul. My blanks would be the wash I used. 

11. I then added 800 ul of ZymoPure Wash 2 

    --> centrifuge 1 minute. Aliquot before dumping. 

    --> Wash 2: 0.3 ng/ul

12. I do another 800 ul of ZymoPure Wash 2 and got 0.7 ng/ul 

13. I centrifuge the tube again to get rid of any excess wash substance. 

14. I the transfer the Zymo-Spin II-PX column into a clean 1.5 mL tube and added 25 ul of Zymopure Elution and incubate it for 2 minutes at room temperature. 

    --> Centrifuge it for 1 minute in a microcentrifuge. 

    --> I finally aliquot the last sample with the elution buffer as my blank. I got 32. 2 ng/ul


Discussion: 

Unfortunately, the sample did not show on a gel which confused my teammates and I because I had a good concentration (32.2 ng/ul). We will discuss some errors that we each have done and why the samples did not show up. 

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