Mutagenesis Series: PCR on Deinococcus radiodurans

By TT -

 Introduction 

PCR is an abbreviation for Polymerase chain reaction, a laboratory technique used to multiply a specific segment of DNA. PCR uses short synthetic DNA fragments called primers to isolate a specific segment of a gene to be amplified.  Then multiple rounds of DNA synthesis are conducted to amplify that segment, producing millions of copies of the specified gene.

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 

Deinococcus radiodurans DNA was extracted using a DNeasy Ultra Clean Microbial Kit.  Then, the extracted DNA was prepped for PCR.  

A total volume of 20 uL was used,  which included: Tube 1: 17 uL of a Q5 master mix, 1 uL of each primer (totaling 2 uL) and 1 uL of the genomic DNA; Tube 2: 14 uL of a Q5 master mix, 2 uL of each primer (totaling 4 uL) and 2 uL of the genomic DNA. Primer 1 and Primer 2 were used for what will be the right fragment of  DNA. Primer 5 and Primer 6 were used for what will be the left fragment of the DNA.  

Once the sample was prepared, PCR was conducted using a thermocycler with an annealing temperature of about 61 degrees Celsius. 

Results 


The fragments were verified through Gel Electrophoresis. The right fragment was successful and was extracted from the gel and cleaned of debris. The left fragment did not show which indicated a failure in the procedure of that fragment. The left fragment had the contents of Tube 1, so there may have been a miscalculation of concentration.  The next step will be to correct this error, the volume measurements from Tube 2 will be used to remake the left fragment to correct this issue. 

References 

Smith, M., Ph.D. (2022). Polymerase Chain Reaction (PCR). National Human Genome Research Institute. https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction

Comments

  1. EricaSeptember 30, 2022 at 5:06 AM

    Best of luck on the next round of PCR for your missing fragment.

    ReplyDelete

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