IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week7/Chemical and Light: Difference between revisions
| Line 211: | Line 211: | ||
===Digestion of P18, P63, and P75 8/4=== | ===Digestion of P18, P63, and P75 8/4=== | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''''' | | align="center" style="background:#f0f0f0;"|'''''' | ||
| Line 232: | Line 231: | ||
| Volume||50 uL||25 uL||25 uL | | Volume||50 uL||25 uL||25 uL | ||
|} | |} | ||
====Gel of Digestion 8/5==== | |||
===Ligation of P18+P63 and P75+P63 8/5=== | ===Ligation of P18+P63 and P75+P63 8/5=== | ||
Revision as of 21:54, 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) | |
HO-pcyA and ompR 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
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.
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.
Expected band sizes are indicated
Last 11
Lanes 1-9: ompR (~750 bp)
Lane 10: P20 colony (1122)
Lane 11: P20 plasmid
Lane 12: 1 kb ladder
Previous 11
Lanes 1-11: ompR (~750 bp)
Lane 12: 1 kb ladder
Previous 11
Lanes 1-11: ompR (~750 bp)
Lane 12: 1 kb ladder
Previous 11
Lane 1: 1 kb ladder
Lanes 2-7: CDF
Lanes 8-12: ompR (~750 bp)
Previous 11
Lane 1: 1 kb ladder
Lanes 2-12: CDF (~900 bp)
Previous 11
Lane 1: 1 kb ladder
Lanes 2-7: HO-pcyA (~1400 bp)
Lanes 8-12: CDF (~900 bp)
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
| ' | P63 | P75b | P18 |
| DNA | 30 uL | 6 uL | 11 uL |
| 100x BSA | 0.5 uL | 0.25 uL | 0.25 uL |
| 10x Buffer | 5 uL Buffer 2 | 2.5 uL Buffer 2 | 2.5 uL Buffer 2 |
| RE 1 | EcoRI 1 uL | EcoRI 1 uL | EcoRI 1 uL |
| RE 2 | SpeI 1 uL | XbaI 1 uL | XbaI 1 uL |
| Water | 12.5 uL | 14.25 uL | 9.25 uL |
| Volume | 50 uL | 25 uL | 25 uL |
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
mtrB, P97, P63 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).
