Contributors: Tank
Purpose
Biological replicate of Run 3, with temperature control added. Test ID 270. Gain 1000, bottom read, 2 min × 80 cycles = 158 min, 37 °C on, triplicates.
Results
| Observation | Value | Meaning |
|---|---|---|
| Plate-warming artefact | Sample rate climbed 26 → 58 FSU/min over the first 60 min while the 50 µM standard fell 9% | Enzyme rate rises with temperature while 4-MU quantum yield falls; the plate took ~80 min to reach 37 °C |
| k at 37 °C | 1,617 FSU/µM vs 1,960–2,060 at room temperature | 18% lower, consistent with standards falling during warming |
| Linear limit, re-tested | 6.25 µM at 99% of the fitted line; 12.5 µM at 95% | Same as Runs 2 and 3 |
| Efficiency (window 80–158 min) | 0.50 → 80%, 0.25 → 82%, 0.125 → 84% | Three points agree to 5%; the 1.00 group is the outlier |
| CV of the 1.00 group | 11.6% (six wells: 47.9–62.8 FSU/min) | Worst on the plate — neat lysate is the most viscous and hardest to pipette accurately |
| Bad standard wells | C8 97% off, E11 21% off | Triplicates saved both by median |
Problems and actions
- Plate wasn’t pre-equilibrated; first 80 min unusable → either pre-warm 30 min, or read to 180 min and fit only the 80–180 min window. The standards themselves report when the plate is at temperature — they stop falling.
- Neat lysate has the worst CV → adopt a diluted working stock going forward.
Cloning side, same day: colony PCR on the Gibson transformation plate
Separately from the MUC work above — Heather’s protocol. Six colonies chosen from the 50 µl Gibson-transformation plate (labelled 1–6); 40 µl of each resuspended in 200 µl LB, 10 µl left in the PCR tube. Colonies 5 and 6 were noticeably smaller. Negative control: no DNA, extra 10 µl water added instead.
Master mix (mm10): 50 µl Taq reaction buffer, 5 µl dNTPs, 2.5 µl for + rev primers (the first number here is overwritten in the original), 1.25 µl GoTaq, 93.75 µl water.
Thermocycler: initial denaturation 95 °C/2 min; 25 cycles of 95 °C/30 s, 55 °C/30 s, 72 °C/2 min 30 s; final extension 72 °C/5 min; held at 4 °C.
New ladder made (20 µl 1 kb ladder + 80 µl loading dye + 100 µl water). Lanes: ladder (10 µl) | 1 | 2 | 3 | 4 | 5 | 6 (25 µl each). Colony 5 ran closest to 2.5 kb, the expected size.
Colonies chosen and error-prone Taq PCR
No date is written on this page. Placed here as a direct continuation of the colony PCR above (same colonies), and because the 4 September Congo red screen tests exactly these same colony numbers the next day.
Colonies chosen: 1, 3, 5, 6 — plated on CMC plates alongside CxnA and CxnA-His. 1 µl of each colony’s 200 µl PCR resuspension was used (colonies 1, 3, 5, 6). CxnA-His was IPTG-induced beforehand (side note). Overnight cultures were spotted on the CxnA / CxnA-His plate, and the rest of each 200 µl resuspended culture went into a fresh overnight culture (10 ml LB + 10 µl 34 mg/ml chloramphenicol), left shaking at 37 °C overnight.
Separately, an error-prone Taq PCR (done by Heather) was loaded on a 0.8% gel: lanes ladder (10 µl) | S | (spacer) | S2 | (spacer) | E | (spacer) | ladder. The ladder looked odd again; S2 (higher MgCl₂ concentration) looked better than S; the error-prone (E) reaction didn’t work.