IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week7/Chemical and Light: Difference between revisions
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[[Image:8-5_cph_p1%2C3_hodgepodge.jpg]] | [[Image:8-5_cph_p1%2C3_hodgepodge.jpg]] | ||
Bands excised and digested: Cph/P105, P3, mtrB | Bands excised and digested: Cph/P105 (AK), P3 (AK), mtrB (XP, ES) | ||
Bands excised and saved: | Bands excised and saved: ompR, HO-pcyA, CDF | ||
=TOPO cloning= | =TOPO cloning= | ||
Revision as of 06:53, 6 August 2008
PCR
8/4: Cph/EnvZ, P1, P3, mtrB not BB
|
1% agarose gel visualized using EtBr/UV | |
|---|---|---|
| Lane | Contents (approx. expected band size) | |
| 1 | 1 kb plus ladder | |
| 2-5 | cph-EnvZ 50 °C annealing temperature (2.3kb) | |
| 6-7 | P1 (3kb) | |
| 8-9 | P3 (3kb) | |
| 10-11 | mtrB (2.1kb) | |
| 12-13 | HO-pcyA (1.4kb) | |
| 14-15 | ompR (750bp) | |
Bands excised, purified, and digested (ApaLI & Kpn overnight).
8/4 Colony PCR of putative mtrB samples
We picked 24 colonies each of old putative P98 and newly transformed putative P98+63 for colony PCR using the BioBrick primers and 55°C annealing for 30 cycles. If the colonies contained mtrB, a band at ~2.1 kb should appear. No colonies appeared to contain mtrB.
All gels with 1kb plus ladder
| P98 | P98 | P98+63 |
 |
 |
|
| P98+63 | Mix | |
 |
 |
Lane 1: 1 kb plus ladder |
8/4 CphEnvZ, P1, P3, mtrB
8/5: gels
Gel for excision (includes old PCR samples)
Bands excised and digested: Cph/P105 (AK), P3 (AK), mtrB (XP, ES)
Bands excised and saved: ompR, HO-pcyA, CDF
TOPO cloning
08/04
P1, mtrB
These had a few white colonies. Colony PCR, however, indicated that the correct PCR product was not incorporated.
ompR
All colonies were blue.
HO-pcyA
No colonies.
Transforming in XL10-Gold cells
| Plate | Marker | # Colonies | Description |
| XL10-Gold P75a + P63b (1:1) | Kan | 2 | No fluor |
| XL10-Gold P75b + P63b (1:1) | Kan | 6 | 1 fluor |
| XL10-Gold P75a + P63b (6:2) | Kan | 0 | |
| XL10-Gold P75b + P63b (6:2) | Kan | 1 | fluor |
| XL10-Gold P75a + P63b (7:1) | Kan | 1 | no fluor |
| XL10-Gold P75b + P63b (7:1) | Kan | 2 | both fluor |
| XL10-Gold Dephos P75a | Kan | 1 | |
| XL10-Gold Dephos p75b | Kan | 0 | |
| XL10-Gold P3 + (P39+p51) 2/6 | Kan | ||
| XL10-Gold P3 Topo w/ XGAL | Amp | ||
| XL10-Gold P1 Topo w/ XGAL | Amp | ||
| XL10-Gold mtrB Topo w/ XGAL | Amp | ||
| XL10-Gold pUC18 | Amp | 100s | White, very small |
| XL10-Gold negative control | Amp | many | extremely small |
| XL10-Gold negative control | Kan | 0 |
Ligations
HO-pcyA, ompR, CDF
Making Thermoinducible cI System
Ligation from 8/1
Plate Results (also listed in Week 6):
| Plate | Marker | # Colonies | Description |
| E1 P75a + P63b (1:1) | Kan | 0 | |
| E1 P75b + P63b (1:1) | Kan | 0 | |
| E1 P75a + P63b (6:2) | Kan | 1 | No Fluor, 3mm |
| E1 P75b + P63b (6:2) | Kan | 1 | Fluorescent, 1mm. |
| E1 P75a + P63b (7:1) | Kan | 0 | |
| E1 P75b + P63b (7:1) | Kan | 0 | |
| Dephos P75b | Kan | 0 |
Fluorescent colony was picked for liq culture and restreaked. Miniprep sent for sequencing-- ligation confirmed by sequencing (8/5).
Analytical Digest Gels from TOPO Cloning 8/5
[Gels here]
Strange bands, but sent several clones for sequencing anyway.
Digestion and Ligation of P18+P63 and P75+P63
Done in tandem and side by side with Christina (except for digestion).
Digestion of P18, P63, and P75 8/4
Gel of Digestion 8/5
Ligation of P18+P63 and P75+P63 8/5
Used dephosphorylated and undephosphorylated vector.
Plate Results 8/6
Digestion and Ligation of P63 + cI857 (Both Primer Sets)
Done with gel purified and pcr purified cI857, and in tandem with Christina.
