Mutagenesis series: Ligation in Higher Volume

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 th...