@@ -152,13 +152,15 @@ After running our purified phi29 DNA polymerase from immobilized metal affinity
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caption='Figure 14. SDS-PAGE with purified phi29 DNA polymerase, displaying bands at 50kD ~ 75kD'/>
Our team tested the efficiency of the purified phi29 DNA polymerase in rolling circle amplification (RCA). We ran the rolling circle products on a gel and saw fluorescent bands of DNA very close to the well, indicating that the RCA reaction was successful (see Fig. 15).
Our team tested the efficiency of the purified phi29 DNA polymerase in rolling circle amplification (RCA) by comparing the rolling circle products (RCP) of the commercial and purified phi29 DNA polymerase. We added the same volume of both to RCA reactions and ran the rolling circle products on a gel. We saw fluorescent bands of DNA very close to the wells, indicating that both the RCA assay utilizing commercial and purified enzyme reactions were successful (see Fig. 15).
If we buy phi29 DNA polymerase from New England Biolabs, it will cost \$251.00 for 1,250 units. However, after we utilized the NEBExpress Ni Spin Columns (\$10 per prep to purify the enzyme), we were able to produce 200ul in one prep, or approximately 2,000 units. Because, it can be assumed that the unit between the commercially bought enzyme and our purified enzyme is similar, if commercially bought, this 200ul amount would cost around \$400. Therefore, we can determine that our purified protein is significantly more affordable than the commercially bought protein.
caption='Figure 15. Gel comparing the rolling circle product (RCP) between the team’s purified phi29 DNA polymerase and that commercially available phi29'/>
However, we were not able to compare the cost of our purified protein with our commercially bought protein as each prep of purification has to be compared individually due to unit differences. In the future, Lambert iGEM hopes to purify phi29 DNA polymerase and compare the cost of our purified protein to commercially bought protein.
In the future, we plan to more accurately quantify the activity of the purified units of phi29 DNA polymerase. Additionally, we hope to test the accuracy of our enzyme by quantifying the RCP using linear DNA probes (see [RCA: outputs](https://2023.igem.wiki/lambert-ga/rca/)) to validate its use of point-of-care testing.
