Kanamycin Resistance Gene-containing Plasmid Extraction from E. coli

Bacterial Plasmid Extraction: A Summary

March 4th, 2022

Introduction

Plasmids are unique circular structures of double-stranded DNA replicating outside of the nuclei of bacterial cells. Because of the nature of plasmids, they can be used in genetic modification of certain microorganisms by inserting genes of interest into the plasmid. The cells containing the plasmids then replicate the inserted gene, and the plasmids can be isolated for the purpose of preserving the genetically modified plasmid genome. In this week's lab, plasmids containing the gene for kanamycin resistance are extracted from Escherichia coli for use in future transformation and Gibson Assembly procedures. It is hypothesized that a sufficient amount of plasmids containing the kanamycin-resistance gene will be collected during this procedure since E. coli grows very well on agar plates with the added kanamycin antibiotic (meaning that almost all E. coli cells grown on the agar plate with kanamycin contain the plasmid). However, the factors that could nullify this hypothesis are the cell density of the E. coli broth culture and volume used to carry out the plasmid extraction. A low cell density or volume of broth culture could yield a low concentration of plasmids.  

Procedure

For this procedure, the Monarch Plasmid Miniprep Kit is used, and the protocol can be summarized in the following steps:

• Using four 2 mL samples of E. coli broth culture, pellet bacterial cells on the bottom of the 2 mL tubes by centrifuging for 30 seconds, discarding supernatant after centrifugation. 
• Resuspend collected pellet in 200 microliters of Plasmid Resuspension Buffer labeled B1. Vortex as needed to homogenize the cells. 
• Add 200 microliters of Plasmid Lysis Buffer labeled B2, but do not vortex in this step. Instead, invert tubes a few times for homogenization, and then incubate them at room temperature for 1 minute. The resulting mixture will look as a transparent, viscous, dark pink solution. 
• Add 400 microliters of Plasmid Neutralization Buffer labeled B3, invert a few times to homogenize, and incubate at room temperature for 2 minutes. The resulting solution will have a yellow precipitate. 
• Centrifuge the lysate solutions for 5 minutes to collect a compact pellet at the bottom of the tubes. Now the supernatant contains the desired plasmids and the pellet contains cell debris. 
• Transfer the supernatant from each tube to spin column tubes using a micropipette without disturbing the pellet. Centrifuge the spin columns for 1 minute and discard the flow-through. This step might need to be repeated if the volume of the supernatant is large. 
• After placing the spin columns back into the tubes, add 200 microliters of Plasmid Wash Buffer 1 and centrifuge the tubes for 1 minute. Discard the flow-through as needed.
• Add 400 microliters of Plasmid Wash Buffer 2 and centrifuge for another minute. Discard the tube with the flow-through. 
• Transfer the spin columns to clean 1.5 mL microfuge tubes without touching the tip of the column on anything.
• Add about 30 microliters of DNA Elution Buffer to the center of the white filter inside the spin column, wait for 1 minute, then centrifuge for 1 minute. Now, there is about 30 microliters yield of plasmid DNA ready for future applications. The plasmid solutions are stored in the freezer only after being measured for absorbance values using 260 nm wavelength. 

Results and Conclusion

The extracted plasmids are measured for concentration using a NanoDrop, and the following OD260 values are collected:

Sample 1: 18.1 (Purity: 1.83 A260/A280)

Sample 2: 34.1 (Purity: 1.74 A260/A280)

Sample 3: 24.4 (Purity: 1.73 A260/A280)

Sample 4: 11.4 (Purity: 2.04 A260/A280)

A good OD260 for the extracted plasmids would have had the absorbance of 150-200. As seen above, the concentration of the double stranded plasmid DNA is extremely low. Therefore, it was hypothesized that the low concentration of plasmid DNA was due to using a diluted starting culture of E. coli used in plasmid extraction. Another possible reason for getting a low OD260 for the plasmid concentration is that not enough volume of broth culture was used to extract the plasmid. As a result, the original hypothesis is rejected, and the plasmid extraction protocol will be repeated using 6 mL of E. coli broth culture instead of 2 mL. This will be done in steps, centrifuging 1 mL of broth culture at a time for 30 seconds. In conclusion, after repeating the plasmid extraction procedure using a bigger volume of broth culture, the yielded plasmids will be used in future applications, most important of which is the transformation of the plasmid containing the kanamycin-resistance gene into Deinococcus radiodurans.