Mutagenesis Series: 2-way Assembly

 Introduction

Previously, step 1 of the Overlap PCR procedure was conducted. There were no primers added to these reactions, which led to a gel that showed no bands of DNA because the fragments were not yet fused together.  They were only amplified.  The next step that will be discussed is called the Final Fusion step of this protocol.  This step should result in a 2-way assembly by fusing together the overhang sections created by the primers that will be added. 

Procedure

This protocol was optimized to eliminate the need for optimizing PCR cycling conditions.  There are no specific temperatures that must be used for the annealing cycle and the temperatures decrease on a gradient of 0.5 degrees Celsius per cycle.  

This step was completed in two reactions.  This step required 22.5 uL of Q5 master mix, 4 uL of the step 1 reaction, 1 uL each of primers 1 and 4 (left fragment, tube1), 1u of primers 3 and 6 (right fragment, tube2), and 21.5 uL of PCR water.  

These two reactions were put into a thermocycler using the touchdown method pictured below, for 42 cycles. 

Results

Gel of Final Fusion Overlap PCR Protocol.  

This gel shows a successful 2-way assembly between the 

right + middle fragments and left + middle fragments. 

It is important to note that there was no PCR clean-up completed before the overlap procedure was followed. Following this protocol, including the touchdown thermocycler process, has led to a successful 2-way assembly of D. radiodurans and the KanR plasmid of E. coli.  The next step of this process will be to attempt a 3-way assembly, left+right+middle. 

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