Contributors: Tank
The design change that fixed the linearity problem seen in Runs 1 and 2.
Purpose and rationale
Replaced the volume series with a CxnA : CxnA-His mixing-ratio series. Test ID 267. Changing volume changes enzyme amount and matrix together; holding total lysate at 10 µl and varying only the CxnA : His ratio keeps protein, turbidity, detergent, and pH constant. Gain 1000, bottom read, 2 min × 528 cycles = 17.6 h, 20 flashes, shaking 300 rpm, temperature off.
Results
| Relative enzyme | Rate (FSU/min) | Efficiency |
|---|---|---|
| 1.00 | 55.0 | 100% |
| 0.50 | 29.7 | 108% |
| 0.25 | 15.7 | 114% |
| 0 (pure His) | −0.34 | — |
Dose linearity restored. Compare with the volume series: Run 1 gave 4% efficiency at the top load and Run 2 gave 42%. The earlier collapse was an artefact of changing volume, not a property of the enzyme.
| Also measured | Value |
|---|---|
| Standard curve, PBS | 3.125 µM → 1,986 FSU/µM; 6.25 → 1,957 (99%) — linear limit confirmed at 6.25 µM |
| Matrix recovery R | 0.95–1.00 → 10 µl of lysate is optically neutral |
| Specific activity, three dilutions | 0.221 / 0.229 / 0.223 mU/mg — constant, as linearity requires |
| Bad standard wells | 4 of 16 (F1 no 4-MU, E1 1.7× high, D11 26% low, C4 23% high) |
Problems and actions
- Temperature was off — the plate ran at room temperature all night → set 37 °C going forward.
- 17.6 h read; standards photobleached 13–16% (10,560 flashes per well) → read 150 min instead.
- 25% failure rate on standard wells → go to triplicates so an outlier can be dropped by median.
Cloning side, same day: gel, new-primer Q5 PCR, gel purification
Separately from the MUC work above:
- 250 ml 1× TAE buffer + 2 g agarose + 12.5 µl gel red. 1× TAE made from 1960 ml water + 40 ml 50× TAE. Ladder: 100 µl water + 20 µl ladder + 80 µl loading dye.
- Lanes: ladder (30 µl) | NEB− | NEB 0.5B [?] | NEB 5% | GO− | GO 0.5B [?] | GO 5% | R− | R 0.5B [?] | R 5% (circled) | ladder (30 µl). 10 µl loading dye, 50 µl loaded. Noted: “Loaded way too much ladder.” R 5%: single band cut out.
- An overnight-culture section on this page is scribbled out and mostly illegible — mentions chloramphenicol, CxnA ×2, CxnA-His ×2, 10 ml LB, and IPTG, but not transcribed beyond that.
- Q5 PCR run with new primers, done by Heather, same conditions as previous runs → annealing time turned out to be incorrect for the new primers (62 °C used instead of 70 °C).
- Gel purification of one product: 0.39 g gel slice → 390 µl membrane binding solution, eluted in 30 µl NFW instead of the usual 50 µl. Nanodrop: 8.820, 3.075, 0.665 (unlabelled triplet, presumably conc./260-280/260-230).
Gibson transformation
No date is written on this notebook page. Placed here because it follows directly from the gel-purified product above, and because the 3 September colony PCR explicitly picks its colonies from the 50 µl plate produced by this transformation.
- Defrost competent cells on ice; take 25 µl competent cells per reaction (a struck-out note originally said to take all the competent cells).
- Labelled TGA and TGB (a word before “TGB” is struck out).
- Add 2.5 µl of Gibson ligation DNA (10%) to each — GA / GB.
- Vortex, then ice for 30 minutes.
- Heat-shock at 42 °C for 30 seconds.
- Back on ice for 2 minutes.
- Add 500 µl SOC media.
- Shaking incubator, 250 rpm, 37 °C, 1 hour.
- Spread 50 µl onto LB-chloramphenicol plates.
- Spin the remaining cells at 6,000 rpm for 1 min (+30 s), remove the media, resuspend in 50 µl SOC, and plate the rest onto LB-chloramphenicol.
- Incubated overnight at 37 °C (this last note was added later, in a different pen).