Mutagenesis Series: 3-way assembly

By TT -

 Introduced 

As it was previously mentioned, this part of the process will be using the 2-way assembly fragments, without clean-up, and adding the missing fragment with the appropriate primers. The procedure that is being followed in the process recommends a clean-up of PCR products, however, the 2-way assembly was successful without a clean-up. The results of this attempt will determine the next step of this series.

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, which were the same as the step of creating the 2-way strand. 

It is important to note that the 2-way assembly fragments have not been through a clean-up so using the Gel Excised samples may be more beneficial.  

Results

Gel of 3-way assembly attempt #1.
No bands are shown.  The attempt was unsuccessful. 

The first attempt at creating a linear strand was unsuccessful.  This gel looks like a previous gel from a prior stage of this experiment that had no primers added to the solution.  The assumption is that the primers were not added to the solution so another attempt will be made and ensuring that primers 1 and 6 are added to the solution.


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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