Week of 2/26

 Introduction

This week, we performed a successful inoculation of D. aquaticus from plate to broth. The broth

was used later in the week for DNA extraction using the Zyppy Plasmid Miniprep Kit.

Procedure

The following steps were followed to inoculate D. aquaticus from plate to broth:


- A flask of appropriate size should already have media in it and be labeled with initials,

date, bacteria name, and type of media. The flask should have been autoclaved and

made ready for inoculation.

- Gather a bunsen burner, an inoculation loop, and a plate.

- Heat the inoculation loop over the bunsen burner flame to sterilize the loop, and let it

cool for about 20 seconds.

- Open the flask and have the lid in between your fingers underneath the loop.

- Swipe the lip of the flask over the bunsen burner flame to sterilize.

- Open the plate just enough to collect one colony with the inoculation loop. Close the

plate.

- Place the loop with the colony into the flask and sweep it around to spread the bacteria.

- Swipe the lip of the flask over the flame again and quickly close it.

- Hold the inoculation loop over the flame to sterilize it.

- You are done, and the inoculated broth is ready for incubation.

Several days later, the following steps were followed to extract DNA from D. aquaticus using the

Zyppy Plasmid Miniprep Kit:

- Pipette a 1 mL sample of your inoculated broth into a Eppendorf tube.

- Centrifuge the tube to create a “pellet,” a visible clump of cells at the bottom of the tube.

- Use a pipette to remove the liquid around the pellet without breaking any cells off of the

lump.

- Resuspend the pellet in 600 μL of TE or TGY medium.

- Add 100μL of 7x Lysis Buffer (Blue) to the eppendorf tube and invert the tube 4-6 to mix.

Proceed within two minutes.

- Add 350μL of cold Neutralization Buffer (Yellow) and thoroughly mix the sample.

- When the neutralization is complete, the sample will turn yellow.

- After the sample turns yellow, invert 3 more times to ensure thorough mixture.

- Centrifuge the sample at 11,000-16,000 x g for 2-4 minutes and transfer the sample nto

the Zymo-Spin IIN column.

- Place this new column into a collective tube and centrifuge the sample again, this time

for only 15 seconds.

- Remove the liquid and replace the column into the collection tube before adding 200μL

of Endo-Wash Buffer to the same column.

- Centrifuge the sample once again, for 30 seconds.

- Add 400μL of Zyppy Wash Buffer to the same column and centrifuge for 1 minute.

- Transfer the column into a 1.5 mL centrifuge tube.

- Add 30 μL of Zyppy Elution Buffer to the column matrix and allow to sit for 1 minute at

room temperature.

- Centrifuge for 30 seconds to elute the DNA sample. You are Done.

Results

Through the inoculation of D. aquaticus and the isolation of the bacteria’s DNA, we were able to

prepare what we needed to run a PCR reaction, which I was not present for. The amplified DNA

from the PCR reaction can be used in the following week for gel electrophoresis.

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