Mutagenesis Series: PCR on Deinococcus radiodurans part 2

By TT -

Introduction 

This is a continuation of the previous lab report. The fragments of Deinococcus radiodurans were amplified via PCR and were verified through Gel Electrophoresis. The right fragment was successfully 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. 

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, the previous reaction included: 17 uL of a Q5 master mix, 1 uL of each primer (totaling 2 uL) and 1 uL of the genomic DNA; This round included: 14 uL of a Q5 master mix, 2 uL of each primer (totaling 4 uL) and 2 uL of the genomic DNA. Primer 5 and Primer 6 were used for what will be the right fragment of  DNA.

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

Results 

Before                                         After

The fragments were verified through Gel Electrophoresis using a 1kb ladder. Correcting the measurement error of the Q5 master mix fixed the issue. The left fragment did show which indicated a successful amplification of that fragment. The next step will be to amplify the KanR gene, from the E. coli, as the middle fragment for this assay. 

Comments

  1. EricaOctober 1, 2022 at 1:46 PM

    In your next report, please indicate what we're looking at in your figures.

    ReplyDelete

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