Mutagenesis Series: PCR - E. Coli KanR - Middle Fragment

By TT -

 Introduction 

Again, in this series, Deinococcus radiodurans and E. coli are amplified to isolate certain genes and block a pathway of D. radiodurans. E. coli is used for its KanR gene, which is responsible for the bacterium's ability to resist the antibiotic kanamycin.  The KanR gene in this assay is used to block a specific gene within D. radiodurans known as ZWF of the pentose phosphate pathway. The blockage of this gene will present opportunities for future studies of D. radiodurans in its behavior and ability to survive under extreme conditions. The insertion of the KanR gene is important because it will be used as an indicator that the DNA has been successfully integrated into the DNA sequence due to D. radiodurans becoming resistant to kanamycin. 

Procedures 

The E.coli KanR plasmid was extracted using a Monarch Plasmid Miniprep Kit. The pressure for this bacteria was added in the broth culture and on the plate before extraction.  This applied pressure was the addition of kanamycin.  After the extraction was completed, the plasmid sample was prepped for PCR.  

A total volume of 20 uL was used,  which included: Tube 1: 10 uL of a Q5 master mix, 1 uL of each primer (totaling 2 uL) and 10 uL of the extracted plasmid; Primer 3 and Primer 4 were used to create the middle fragment.  

Once the sample was prepared, PCR was conducted using a thermocycler with an annealing temperature of about 59 degrees Celsius. 

Results 



The fragments were verified through Gel Electrophoresis. The middle fragment was successful and was extracted from the gel and cleaned of debris. The gel extracted samples that have been cleaned will be used for the Overlap PCR Process to create a 2-way assembly of right-middle and left-middle fragments. The right-middle will consist of D. radiodurans and the KanR plasmid from E. coli.

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