Mutagenesis series: Amplification of a linear strand

By TT -

Introduction

DNA ligation was successful. D. radiodurans DNA has been joined together with the DNA of E. coli, specifically the kanR gene, which has specific primers designed to block the ZWF gene. It was suggested to attempt amplification of this linear strand via PCR. 


Procedure

A total volume of 20 uL was used containing: 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 6 were used.  

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

Results 



This attempt was unsuccessful.  The picture above depicts the gel that was run on the sample after PCR and there are no DNA bands shown.  There was 5 uL of DNA used as this amount was used previously when the ligation was successful, so a light band was expected to be seen on the gel. The assumption is that because the ligation was completed under certain conditions that this amplification process will need to be modified to fit those specific conditions. 

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