Site-directed Mutagenesis
- On Friday, we ran Thursday's PCR products on a 1.8% Agarose gel, run at 60V for 3 hours. The samples were loaded as follows:
- 100bp ladder
- Non-primered set without template
- Upstream fragment without template
- Primered set without template
- Downstream fragment without template
- 100 bp ladder
- Downstream fragment
- Primered set
- Upstream fragment
- Non-primered set
- Positive control
- 100 bp ladder
- All other lanes are replications of primered set
- Results:
- Upper lane, wells 1-5 (no DNA set)
- Conclusions:
- Positive and negative controls (no DNA set) indicate effective and uncontaminated PRC reagents
- Our primered set was set between the upstream and downstream section for comparison. Unfortunately, this was the only sample that did not appear. We may have forgot the template DNA. However, the replicates did appear, and are the same setup. Although we cannot see a direct side-by-side comparison, it appears that we are within 50bp again, as we seen on Thursday. In fact, these look cleaner, but otherwise identical to the original reaction, indicating consistency.
- When comparing relative location to the ladder, the primered set looks very similar to the downstream fragment. In our gel, there are two very faint bands at ~350bp and ~450bp for the downstream fragment. They are brighter in combined primer set. This is likely the same profile but at different amplifications.
- The non-primered set did not work again, as a matter of verification.
- This gel shows a much cleaner result of our work so far. It seems that we amplified the two fragments correctly, but we will need to modify our approach peice our fragments together.
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