Mutagenesis series: 3-way assembly pt 2

By TT -

 Introduced 

The first attempt at creating a linear strand was unsuccessful.  This gel looked 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 was made and  primers 1 and 6 were added to the solution.


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. 


Results

Gel of 3-way assembly attempt #2.
Bands shown are about 2300 base pairs.  
The attempt was successful. 

The second attempt at creating a linear strand was successful.  The band is very light but it is the correct size, compared to the 1kb ladder and only 5uL was used in the dye solution. The next step is to amplify this fragment to transform D. radiodurans cells 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

Comments

  1. Genaro RiveraDecember 9, 2022 at 9:21 PM

    I like how you explained the procedure, but I would like to see how you came up with this solution

    ReplyDelete

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