Blog#4 2/5/23 - 2/16/23

 Introduction

The previous week, I researched different chemical tests that help characterize bacteria. These tests range from the bacteria's physical features to reactions to growing in different media. We also had a discussion about the Amc cycle. The discussion was a surface explanation of the cycle. I also learned how to make double TGY  and R2A media. I also learn the steps of RNA isolation. the following Friday, I assisted Madison as we attended to an RNA isolation of E. coli. 

Methods 

Characteristic tests

First, you can assess with your eyes anything particular about the appearance of the bacteria, the way the colonies look, its edges, and things you think are worth mentioning. There is the gram-stain test to figure out if a bacteria is gram-negative or gram-positive. after the stain, you can look at the bacteria under the microscope to figure out the bacteria's arrangement and morphology. You can try to grow them in selective and differential media, these media can determine this like if the bacteria can fermentate lactose or sugar. 

Rna isolation

1. Resuspend fresh or frozen call pallet in 800 RNA LY Lysis Buffer and Fansder the mature ZR BashingBead Lysis Tube

2. Secure the tube in a bread beater fitted with a 2 ml tube hold assembly and process

3. Centrifuge the tube for 1 minute to pellet debris

4. Transfer up το 400 μ of the clear supernatant into Zymo-Spin ICQ Colume in a t Collection Tube and centrifuge. Save the flow through

5. To the flow through add an equal volume of ethanol (95-100%) (1:1) and mix well

6. Transfer the mixture into a Zymo-Spin UCR Column in a Collection Tube and centrifuge. Discard the flow-through.

7. Add 400 µl RNA Prep Buffer to the column and centrifuge. Discard the flow-through

8. Add 700 µl RNA Wash Buffer to the column and centrifuge Discard the flow-through.

9. Add 400 RNA Wash Butter and centrifuge the column for 1 minute to ensure complete removal of the wash buffer. Then carefully, transfer the column into a nuclease-free tube 

10. Add 50 µl DNase/RNase-Free Water directly to the column matrix and centrifuge

To make double TGY you follow the same measurements of regular tgy but you double the amount of tryptone you add. Then to make R2A media you just have to mix R2A, deionized water, and agar. 

Results

The results of the RNA isolation were not good, we were not able to complete the process

the results of Nanodropping 

A- 0.16

B- 1.06

C- 0.09 ( after one and a half hours of incubation)

Discussion and conclusion

I assisted Madison while she did the RNA isolation. I handed her the things she needed and put the tube in the centrifuge. A mistake was made in step 7, instead of the buffer, RNA lysis was added to the tube. This caused our results to turn out badly. The mistake was one that anyone could make, next time we can just proceed with more caution as to what is going into our tube. In the following week, we will attempt again with the RNA isolation. We also learn how to use the fluorescent microscope, along with going further into detail about the AMC cycle. 

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