Mutagenesis series: Ligation in Higher Volume

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 Introduced 

The previous attempt to amplify a linear strand was unsuccessful.  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. In this attempt, the Overlap process was used to remake the linear strand.  There were 6 tubes used to make enough of the sample to use for transformation. 


Procedure 

Using the 2-way assembly of the right+middle, the addition of the left fragment was introduced to the solution that consisted of a 5X Q5 master mix, primers 1 and 6, and PCR water.  This process went through Overlap PCR and the Final Fusion steps of the Overlap PCR protocol.  The conditions of the thermocycler were set to the prescribed temperatures and durations that are detailed in the Overlap PCR protocol.  The same mix was used for all 6 tubes.


Results


Gel of 3-way assembly.
Bands shown are about 2300 base pairs.  
The attempt was successful. 

The second attempt at creating a linear strand  with more volume was successful.  The band in lane 4 is very light but it is the correct size, compared to the 1kb ladder and only 5uL was used in the dye solution for each sample. The next step is to make competent cells of D. radiodurans to transform to be resistant to kanamycin which is blocking the ZWF gene which is a pathway to glycolysis. 


References

Hilgarth, R. S., & Lanigan, T. M. (2019). Optimization of overlap extension PCR for efficient transgene construction. Elsevier. https://doi.org/10.1016/j.mex.2019.12.001

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