Mutagenesis series: 3-way assembly planning

By TT -

Introduced 

The next step of this process will be to attempt a 3-way assembly, by combining left+right+middle fragments. This process will create a linear strand of DNA that will be transformed and taken up by competent Deinococcus radiodurans.   

Procedure

There are 2 options that may work in the assembly of a 3-way linear strand of DNA.  

1.) Using the 2-way assembly of the right+middle or left+middle would allow the addition of just one fragment to the solution such as adding the left fragment to the right+middle assembly.  This process will go through Overlap PCR and the Final Fusion step, to attempt to make the linear strand.

2.) Using the original individual fragments and placing them in one tube. This would also require Overlap PCR and the Final Fusion step.  

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.  The concentration of these samples is very low so it may need to be concentrated before it can be used for Overlap PCR to make a 3-way linear strand. 

The primers that are required for this procedure are primer 1 and primer 6. 

Results

The first attempt at this process will be to use the 2-way assembly fragments, without clean-up, and add 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.


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

Comments

  1. EricaNovember 4, 2022 at 8:34 AM

    Nice work this week. Hope you have success with your ligation!

    ReplyDelete

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