IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week7/Chemical and Light: Difference between revisions
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Revision as of 17:34, 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
All three ligations yielded >50 colonies on the TOP10 cell plate with a 5 minute ligation in all combinations (HO-pcyA and ompR with P1 and old P3; CDF with P3). The P90 (CDF+P3) did not fluoresce. The DH5α cells did not have nearly as many (<5) and ligation times (10min, 30 min) did not appear to be significant.
Colony PCR
We picked at least 18 colonies from each ligation set, patched a master plate, inoculated liquid cultures, and performed 12.5 μL colony PCR reactions. The BBp/sfx primer pair was used, annealing temperature was 50°C for first 10 cycles and 54°C for rest of the cycles. 2 controls were performed: one using a picked P20 colony, and another with miniprepped P20 plasmid.
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.
Digestion of cI857 PCR product and P63 8/5
RE digests 08/06
We digested mtrB with ES and XP so that we can put it into the P63 (terminator) and P97 (RBS) vectors. We also digested P97 with SP (and dephosphorylated it).
Ligations and transformations 08/06
We ligated P105 and P1/P3 not BB, and mtrB not BB and P1/P3 not BB. We tried letting the ligation reactions run for 5 and 10 minutes. We used P1/P3 not BB from 08/05 and from another date (older). We transformed using TOP10 and DH5α. We also retransformed P108, the Lac QPI with a high constitutive promoter in a p15A vector.
Colony PCR 08/06
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