Week of 08/26/24

 


Introduction

Plasmid extractions are performed upon E. coli cells containing the PRAD1 plasmid that contains the genes for both chloramphenicol and ampicillin resistance. E. coli is easy to grow and is thus a useful vector for the PRAD1 plasmid. The plasmid can be replicated and stored by maintaining E. coli colonies on LB plates containing ampicillin. The ampicillin is necessary within the LB plates to maintain an environmental pressure for the E. coli to hold onto the PRAD1 gene. Without this environmental pressure, E. coli would eject the PRAD1 gene as bacterial cells are extremely energy efficient and do not hold onto genes that are not necessary for survival. 

A zyppy kit DNA retrieval protocol (and kit) can be used to lyse cells and extract the plasmid DNA within. The extracted plasmid can be used in a transformation procedure. The plasmid can potentially be inserted into a new species which would then allow for the genetic expression of the plasmid DNA within the new species. The goal here is to have the transformed species express resistance to ampicillin and chloramphenicol as a sort of “proof of concept” to prove that the species is indeed transformable.

After a zyppy kit plasmid extraction is performed, a gel electrophoresis procedure is done to confirm that the PRAD1 plasmid has been correctly isolated. A gel is also run after a possible transformation to confirm that the PRAD1 plasmid is within a new species. 


Methods

Zyppy kit plasmid extraction protocol

Optional First Step 

Pack Cells by centrifuging a few times (removing supernatant and adding more sample broth after each centrifuge)


1) 600 microliters of bacterial culture grown in LB to 1.5 ml microcentrifuge tube

2) Add 100 microliters 7x lysis buffer (blue). Mix by inversion 4-6 times.

3) Add 350 microliters of cold neutralization (yellow). Invert sample 2-3 times.

4) Centrifuge 11000-16000x g 2-4 minutes

5) Transfer 900 microliters supernatant into provided zymo-spin IIN column. Don’t disturb cell debris pellet

6) Place column into a collection tube. Centrifuge for 15 seconds.

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

8) Add 200 microliters of endo-wash buffer to the column. Centrifuge 30 seconds. 

9) Add 400 microliters of zyppy wash buffer. Centrifuge for 1 minute

10) Transfer column into a clean 1.5 ml microcentrifuge then add 30 microliters of zyppy elution buffer into column matrix. Let stand one minute.

11) Centrifuge for 30 seconds to elute plasmid DNA


Transformation Protocol

1) 1 ml cells OD value of 1 (late log)

2) Pellet

3) Resuspend in 100 microliters of TGY

4) Add 40 microliters of 0.3 CaCl2 mix

5) Ice 15 minutes

6) 30 microliter sample and about 10 microliters of plasmid (where the plasmid is 200-400 ng per microliter for a grand total of around 1 microgram of plasmid DNA)

7) Mix

8) Incubate at 30 degrees Celsius for 90 minutes while inverting every 15 minutes.

9) Add  2 ml of TGY and Incubate for 24 hours

10) Plate cells on TGYA and TGYA CM plates

11) Use Cells mixed with PCR water as a blank


Gel Electrophoresis Protocol

1) 30ml of TAE buffer in a small flask

2) Add 0.3g of gel red agarose

3) Cover with napkin and microwave for 30 seconds. Stir and microwave for another 30 seconds

4) Let cool for 5 minutes

5) Pour into a gel apparatus (2 blocks, carrier, teeth)

6) Let solidify

7) Remove sliders and cover with TAE buffer

8) Insert DNA into wells with a 6 microliter ladder combined with 2 microliters of dye alongside samples consisting of: 2 microliters of DNA, 2 microliters of dye and 8 microliters of PCR water

9) Run the gel at 75 mA* from black to red

*55mA for bigger DNA strands

Results

Extracted plasmids with concentrations of 202.3 and 220.4 ng/ microliters. Ran a gel to confirm PRAD1 extraction (the image above).

Discussion

The extraction was a complete success! Concentrations of plasmids are usually much lower so we extracted a great stock. This was Riyah’s and Andrew’s first plasmid extraction and I am very happy with their results. 

Unfortunately, the gel electrophoresis came out messy. It turns out that the dye was loaded into the space for the ladder and the ladder was loaded into the space for the dye. It goes to show just how important it is to correctly decipher the labels as we prepare experiments. Fortunately, after identifying this mistake we were able to confirm that the PRAD1 plasmid was extracted. 


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