Lab Notebook

12 September 2025

• Checked equipment and 1 kb plus ladder with gel electrophoresis protocol.
• Gel appeared as expected.

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13 September 2025

• Carried out RPA protocol (5 identical trials) using 6 bp overlap primer and 10 ng mutation input.
• Observed bands around 200 bp (expected amplicons), 300 bp (template length), and 500 bp (unexplained).

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14 September 2025 — Checking Other Primers

• Carried out 2 identical RPA trials with 4 bp, 5 bp, and 8 bp overlap primers using mutation input.
• 5 bp overlap primer appeared most promising, showing the brightest amplicon band slightly below 200 bp.
• The 500 bp band persisted.

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16 September 2025

• Ran RPA protocol with wildtype, mutation input reactions, and a negative control for 4 bp, 5 bp, and 8 bp overlap primers.
• Input was 1 ng to confirm whether the 300 bp band represented input templates.

Gel layout:

1. DNA ladder
2. 4 bp Overlap Primer + wildtype input
3. 4 bp Overlap Primer + mutation input
4. 4 bp Overlap Primer + no input
5. 5 bp Overlap Primer + wildtype input
6. 5 bp Overlap Primer + mutation input
7. 5 bp Overlap Primer + no input
8. 8 bp Overlap Primer + wildtype input
9. 8 bp Overlap Primer + mutation input
10. 8 bp Overlap Primer + no input

Observations:

• Expected-size amplicon still shows brightest band in the mutation input reaction.
• 500 bp band still present.
• 300 bp band disappeared → likely the input template (1 ng too faint for SYBR Safe).
• Wildtype shows positive results, suggesting low specificity.
• Negative control positive, possibly due to contamination.

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23 September 2025

To check for contamination and improve specificity:

• RPA protocol repeated with only 5 bp overlap forward primer (best in dry lab).
• Shaking incubation (300 rpm) introduced to improve specificity.
• Reactions run with varying input DNA concentrations to assess input effect.
• PureLink PCR Purification Kit used to purify amplicons and remove protein–DNA complexes.
• Input DNA (template + primer pairs) and concentrated templates were also loaded for comparison.

Sample order:

1. 100–300 bp DNA ladder
2. RPA (0 ng mutation DNA input)
3. RPA (0.5 ng mutation DNA input)
4. RPA (1 ng mutation DNA input)
5. RPA (5 ng mutation DNA input)
6. DNA input (5 bp overlap forward + reverse primer + mutation DNA)
7. 100 ng wildtype DNA template
8. 100 ng mutated DNA template

Discussion Points:

1. Why is there a 180 bp band in the negative control? → possible primer dimer.
2. What is the 500 bp band seen only in mutation samples?
3. Why do two bands appear in the template stock solution?

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24–25 September 2025

To purify template inputs and troubleshoot primer design, PCR was run for all primers using mutated, wildtype, and negative (water) controls.

Gel layout (left → right): Each group: same primer pair + (wildtype → mutation → negative control). Groups differ by primer type and annealing temperature:

1. Wildtype forward primer + reverse primer (72°C)
2. 4 bp overlap primer + reverse primer (72°C)
3. 5 bp overlap primer + reverse primer (71°C)
4. 6 bp overlap primer + reverse primer (70°C)
5. 8 bp overlap primer + reverse primer (72°C)
6. Forward template primer + reverse template primer (61°C)

Conclusions:

• Even with PCR, none of the primers reached the desired specificity.
• All primers amplified mutation templates cleanly, confirming primer design validity.
• Further processing is required after amplification to distinguish wildtype from mutation before LFA readout.

To ensure primer performance and template purity:

• PCR-amplified input templates and 5 bp amplified samples were run at high input concentrations.
• Bands were purified using the PureLink Quick Gel Extraction Kit.
• Sanger sequencing was conducted to verify the amplified material.

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(iGEM Meeting Notes)[https://static.igem.wiki/teams/5601/documents/igem-meeting-notes.pdf]