Blog #13 4/22/24 - 4/26/24

Introduction 

This following week I changed groups and joined Anh and Jayce. I learned about their project and some of the major processes they use frequently. These processes are creating competent cells, plasmid extraction, and transformation. We did some inoculations with the transformed cells on agar plates that contained antibiotics.

Methods 

Competent cells

1.) Pipette 1ml of bacteria culture broth, with a Cell density tube Of 109 into a sterile microcentrifuge

2.) Centrifuge for 1 minute using the Centrifuge at 12,008 ram after which a is pose of the supernatant

3.) Resuspend the pellet in the following mixture.

-TGY broth:12901 μι

-1m CaCi 2: 51.6 μι

-DI water: 335.1 μι

-cilcerol 60%: 323 μι

 4.) Mix well and place the solution to a 2mL cryovial 

5.) Store in -80°c freezer

Transformation 

1.) aliquot 100 µl Component cells into mircocentrifuge tubes

2.) Add 1μι or < 10 µl plasmid solution to the tube

3.) Place on Ice for 15 min

4.) Place in the incubator (30-32°c) for 45 min agitating every 15 minutes by inverting tubes.

5.) Transfer Content into a 15ml Centrifuge tube w/4 mL Tgy

6.) Incubate in a shaking incubator at 30°c for 16 hours 

7.) Plate onto TGY agar w/ antibiotic 

8.) Observe growth on the plate after overnight -4 days

Plasmid extraction 

1.) Ada 600~1 of bacterial culture to a 1.5 mi centrifuge tube

2.) Add 10011 of 7x lysis buffer (Blue) & mix by inverting. the tube 4-6 times

-Proceed to step 3 w/in 2 minutes.

-after the addition of 7x lysis buffer the solution should change from opaque to clear blue, indicating complete lysis 

3.) Add 350~1 of cold Neviralization butter (yellow) 5 Mix thoroughly 

-The sample will turn yellow when the neutralization is complete

- invert the sample an additional 2-3 times to ensure. 

4.) centrifuge at 11,000-10,000 xg for 2-4 minutes.

5.) Transfer the supernatant (~900 ~ 1) into the provided Zymo spin in the column (avoid disturbing the pellet)

6.) Place the column into a collection tube & centrifuge for 15 seconds

7.) Discard the flow through & place the column back into the same collection tube

8.) Add 200μι  of Endo wash butter to the column centrifuge for t minute 30 seconds

9.) Add 400 al of Zyppy wash butter to the column centrifuge for I minute

10.) Transfer the column into a clean is mi microcentrifuge for ¾  tube then add 30μι

of zippy elution buffer directly to the column matrix & let stand for one minute at room temperature

11.) Centrifuge for 30 seconds to elute the plasmid DNA • Nanodrop

Discussions 

The purpose of the project is to transform D. caeni to be resistant to a specific antibiotic. This gene for antibiotic resistance already exists in E. coli. The plan is to extract a plasmid from E. coli and then attempt to transform Caeni cells to become resistant. To verify we will plate the transformed cells onto agar plates that contain antibiotics and absorb if they grow.