Lab Notebook
Resuspended DNA for Stock Solution (7/30/2025)
Resuspended DNA in appropriate buffer for stock solution [nuclease-free Tris-EDTA, pH 8.0, or 10 mM Tris-HCl, pH 8.0].
- Pulse-centrifuged new DNA before suspension.
- Target concentration: 10 ng/µl.
- 1,403 ng resuspended in ~140 µl.
Transformations — TOP10 & DH5α (7/30/2025)
Prepare aliquots for transformation:
- 25 µl TOP10 with 2 µl synthetic ZnuABC / clonal vector (7/30/25)
- 25 µl DH5α with 2 µl synthetic ZnuABC / clonal vector (7/30/25)
- 25 µl TOP10 with 2 µl pUC19 (T10 on top) (7/30/25)
- 25 µl DH5α with 2 µl pUC19 (7/30/25)
Perform transformation:
- Ice: 15 minutes.
- Heat shock: 42 °C for 1 minute (water bath).
- Return to ice: 5 minutes.
- Add 100 µl SOC (≈4× cell volume for recovery media).
- 37 °C incubation: 45 minutes.
- Plate and spread with glass beads.
- Incubate at 37 °C; check next morning.
Transformation — DH5α Retry (8/2/2025)
Prepare aliquot for transformation:
- 25 µl DH5α with 2 µl synthetic ZnuABC / clonal vector (8/2/25)
Perform transformation:
- Ice: 15 minutes.
- Heat shock: 42 °C for 1 minute (water bath).
- Return to ice: 5 minutes.
- Add 100 µl SOC (≈4× cell volume).
- 37 °C incubation: 45 minutes.
- Plate and spread with glass beads.
- Incubate at 37 °C; check next morning.
Transformations — BL21 (8/4/2025)
Used NanoDrop to measure DNA concentration; tested two plasmid input volumes for BL21 transformations.
Prepare aliquots for transformation:
-
BL21 with synthetic ZnuABC (8/4/2025)
- Volume: 1 µl plasmid DNA
- Concentration: 4 ng/µl
-
BL21 with synthetic ZnuABC (8/4/2025)
- Volume: 10.5 µl plasmid DNA
- Concentration: 4 ng/µl
Perform transformation:
- Ice: 15 minutes.
- Heat shock: 42 °C for 1 minute (water bath).
- Return to ice: 5 minutes.
- Add 100 µl SOC (≈4× cell volume).
- 37 °C incubation: 45 minutes.
- Plate and spread with glass beads.
- Incubate at 37 °C; check next morning.
