Test

The first round of cloning did not produce successful colonies. Due to this issue, the minipreps used were evaluated. Troubleshooting involved testing different spin column tubes to identify those which provided the product with the highest plasmid concentration. Minipreps were then validated using agarose gels. However, even after the ideal spin columns were identified and yields were improved, there was still contamination of the samples. First, the contamination was believed to be genomic DNA and thus, the protocol was adapted to limit its impact. This resulted in no change. Instead RNA contamination was expected to be the problem. To test this, the same minipreps were incubated with RNAse and were re-run on agarose gels alongside their contaminated counterparts. The result of one of these gels can be seen below:

The RNAse treatment was effective, as can be understood by the lack of RNA contamination seen in the last three lanes. The miniprep protocol was adapted to ensure adequate RNAse addition.

Agarose gels were also used to visualize the efficiency of different restriction digest enzymes using various samples and controls to mimic fully, partially, or incomplete digestion reactions. We compared Thermofisher FastDigest enzymes with NEB-HF enzymes to find those that functioned best under the desired conditions. Gels indicating the conditions that were tested are shown below:

The lanes after the ladder in display double restriction digests using a NotI FastDigest from Thermofisher and EcoRI-HF from. Under these conditions, it would be expected that the enzymes be capable of completely digesting the circular plasmid into a smaller linearized empty backbone fragment. However, in these lanes, roughly three bands can be seen, demonstrating that the plasmid is not being completely digested. Instead, some is remaining circular and some has solely been cut by one enzyme. To address this, we wanted to compare the efficiency of using two thermofisher enzymes. The results of this digestion are seen below. We used the pUCIDT-16s backbones instead as the differences in band sizes were easier to observe.

In this case, digestion appears to have been completed much more successfully as the bands locating the circular plasmids and single cut linear backbones are either very faint (ex. Double digest pUC-IDT-16s-EC in lane 2) or not present (ex. pUC-IDT-16s-SA in lane 5). This experiment helped us realize that changing the enzymes we use to those from Thermofisher may benefit our results in future restriction digests.

Once restriction digest reactions were successfully tested, the workflow progressed through ligation and transformation. Colony PCR was used to validate successful colonies but continuously failed. First, insert specific primers were used but allowed for non specific DNA amplification as can be seen in the following image:

Next, backbone specific primers were attempted, although no amplification of the insert region was seen. Refer to the image below for an example:

Various tests were carried out to modify the annealing temperature, both for reactions at constant temperature and over temperature gradients, to include and exclude the primer’s leader sequence. However, even with these changes little amplification was seen. It was expected that perhaps the annealing temperatures of the primers were too far apart to prove effective as they differed by slightly more than the recommended 5℃ [1]. Therefore, a new method to test for successfully transformed colonies had to be implemented. Diagnostic agarose gels were run to compare the products of a single enzyme digestion using both pESC backbones and mini-prepped transformants as templates. This enabled improved identification of successful constructs as differing band sizes (due to a lack or presence of the insert 16s DNA) could be visualized. Refer to the results page to view these gels. Sequencing was used to verify that the assemblies were indeed successful.