IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week7/Chemical and Light: Difference between revisions
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=Ligations 08/07= | =Ligations 08/07= | ||
*We ligated P17 in a p15A vector (P1 or P3) to P38 and P39. We used a vector to insert ratio of 1:7. We also prepared a ligation reaction with 1 μL vector (P17) and 7 μL water as a transformation control. We transformed 50 μL TOP10 cells with 7 μL DNA and 100 μL DH5α cells with 7 μL DNA. We also used 7 μL of the vector-only ligation to transform 7 μL DH5α cells. | |||
*To test the Lac QPI system, we ligated P45 (RBS+GFP+terminator) to P108. We used a vector to insert ratio of 1:7. We also prepared a ligation reaction with 1 μL vector (P108) and 7 μL water as a transformation control. We transformed 100 μL DH5α cells with 7 μL DNA and we also used 7 μL of the vector-only ligation to transform 7 μL DH5α cells. | |||
Revision as of 11:38, 8 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
Pl05 (CphEnvZ w/ PstI site mutated out) conditions: 5min @ 94°C → 40x [30s @ 94°C → 30s @ (50.8, 51.5, 52.6, 53.9, 55.4, 56.7, 57.7, 58.3)°C → 2:30 @ 72°C] → 7min @ 72°C → ∞ @ 4°C
8/5: gels
|
1.2% agarose E-gel visualized using EtBr/UV | |
|---|---|---|
| Lane | Contents (approx. expected band size) | |
| 1-8 | P105 (50.8-58.3 annealing temp, increases LTR) - 2.2kb | |
| 9 | 1kb plus ladder | |
| 10 | P1 not BB - 3kb | |
| 11 | P3 not BB - 3kb | |
| 12 | mtrB not BB - 2.1kb | |
|
Gel for excision (includes old PCR samples) | |
|---|---|---|
| Lane | Contents (approx. expected band size) | |
| 1, 15 | 1kb plus ladder | |
| 2-3 | P105 PCR product | |
| 4-6 | P3 PCR product | |
| 7-9 | mtrB not BB PCR product | |
| 10 | P1 PCR product | |
| 11-12 | ompR PCR product (720bp) | |
| 13 | HO-pcyA (1.4kb) | |
| 14 | CDF PCR product (900bp) | |
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.
Digests, 1-8
We ran the digests to see if PCR mutations could have introduced an internal cut site. However, even with the small amount of purified digest we had left, we could see a properly sized band for each of P1, ompR, and HO-pcyA PCR products.
|
1% agarose gel visualized using EtBr/UV | |
|---|---|---|
| Lane | Contents (approx. expected band size) | |
| 1 | 1 kb plus ladder | |
| 2 | Old P1 PCR product AK digested (3kb) | |
| 3 | ompR PCR product AK digested (720bp) | |
| 4 | HO-pcyA PCR product AK digested (1.4kb) | |
| 5-12 | HO-pcyA + P1 ligation colony PCR (1.6kb) | |
9-19
|
1% agarose gel visualized using EtBr/UV | |
|---|---|---|
| Lane | Contents (approx. expected band size) | |
| 1 | 1 kb plus ladder | |
| 2-11 | HO-pcyA + P1 ligation colony PCR (1.6kb) | |
| 12 | HO-pcyA + P3 ligation colony PCR (1.6kb) | |
20-30
|
1% agarose gel visualized using EtBr/UV | |
|---|---|---|
| Lane | Contents (approx. expected band size) | |
| 1 | 1 kb plus ladder | |
| 2-12 | HO-pcyA + P3 ligation colony PCR (1.6kb) | |
31-41
|
1% agarose gel visualized using EtBr/UV | |
|---|---|---|
| Lane | Contents (approx. expected band size) | |
| 1 | 1 kb plus ladder | |
| 2-7 | HO-pcyA + P3 ligation colony PCR (1.6kb) | |
| 8-12 | CDF + P3 ligation colony PCR (900bp) | |
42-52
|
1% agarose gel visualized using EtBr/UV | |
|---|---|---|
| Lane | Contents (approx. expected band size) | |
| 1 | 1 kb plus ladder | |
| 2-12 | CDF + P3 ligation colony PCR (900bp) | |
53-63
|
1% agarose gel visualized using EtBr/UV | |
|---|---|---|
| Lane | Contents (approx. expected band size) | |
| 1 | 1 kb plus ladder | |
| 2-7 | CDF + P3 ligation colony PCR (900bp) | |
| 8-12 | ompR + P1 ligation colony PCR (1kb) | |
64-74
|
1% agarose gel visualized using EtBr/UV | |
|---|---|---|
| Lane | Contents (approx. expected band size) | |
| 1-11 | ompR + P1 ligation colony PCR (1kb) | |
| 12 | 1 kb plus ladder | |
75-85
|
1% agarose gel visualized using EtBr/UV | |
|---|---|---|
| Lane | Contents (approx. expected band size) | |
| 1-2 | ompR + P1 ligation colony PCR (1kb) | |
| 3-11 | ompR + P3 ligation colony PCR (1kb) | |
| 12 | 1 kb plus ladder | |
86-94, P20 controls
|
1% agarose gel visualized using EtBr/UV | |
|---|---|---|
| Lane | Contents (approx. expected band size) | |
| 1-9 | ompR + P3 ligation colony PCR (1kb) | |
| 10 | P20 colony PCR (1122) | |
| 11 | P20 plasmid PCR | |
| 12 | 1 kb plus ladder | |
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).
Ligation transformation results 08/07
| Plasmid | Ligation time | Ligation ratio (vector:insert) | Strain | No. of Colonies |
| mtrB not BB+P1 not BB old | 10 min | 2:6 | DH5-alpha | 1 |
| mtrB not BB+P1 not BB | 10 min | 2:6 | DH5-alpha | 4 |
| mtrB not BB+P1 not BB | 5 min | 2:6 | TOP10 | 88 |
| mtrB not BB+P3 not BB old | 10 min | 2:6 | DH5-alpha | 0 |
| mtrB not BB+P3 not BB | 10 min | 2:6 | DH5-alpha | 8 |
| mtrB not BB+P3 not BB | 5 min | 2:6 | TOP10 | 40 |
| mtrB+P63 | 10 min | 2:6 | TOP10 | 48 |
| mtrB+P63 | 10 min | 1:7 | DH5-alpha | 5 |
| mtrB+P97 | 10 min | 2:6 | DH5-alpha | TMTC |
| mtrB+P97 | 10 min | 1:7 | DH5-alpha | 200 |
| P105+P1 not BB old | 10 min | 2:6 | DH5-alpha | 5 |
| P105+P1 not BB | 10 min | 2:6 | DH5-alpha | 1 |
| P105+P1 not BB | 5 min | 2:6 | TOP10 | 2 |
| P105+P3 not BB old | 10 min | 2:6 | DH5-alpha | 1 |
| P105+P3 not BB | 10 min | 2:6 | DH5-alpha | 8 |
| P105+P3 not BB | 5 min | 2:6 | TOP10 | 55 |
Colony PCR
Ladders (end of each block) alternate as low range (100, 200, 400, 800, 2000bp) and 1kb plus. Expected band sizes indicated.
1-16
Lane 1: Plasmid control P85 (2.3kb)
Lane 2: Colony control P88 (2.2kb)
Lanes 3-16: P105+P3 ligation colony PCR (2.5kb)
4, 6, 14 look like they have potentially correct products, so liquid cultures were grown up.
17-24: P105+P1 ligation colony PCR (2.5kb)
None appear to be correct.
25-48: mtrB+P63 ligation colony PCR (2.2kb)
27, 28 picked.
49-64: mtrB+P97 ligation colony PCR (2.1kb)
51 picked.
65-80: mtrB'+P3 ligation colony PCR (2.3kb)
65, 69, 70, 78, 79, 80 picked.
81-96: mtrB'+P1 ligation colony PCR (2.3kb)
82, 87, 95 picked.
RE digests 08/07
Lane 1: 1 kb ladder
Lane 2: E1P1+P17A cut EX (3652)
Lane 3: E1P1+P17C cut EX (3652)
Lane 4: E1P1+P17D cut EX (3652)
Lane 5: E2P1+P17A cut EX (3652)
Lane 6: E2P1+P17B cut EX (3652)
Lane 7: P108 cut SP (4155)
Lane 8: P45 cut XP (876; 2079)
Ligations 08/07
- We ligated P17 in a p15A vector (P1 or P3) to P38 and P39. We used a vector to insert ratio of 1:7. We also prepared a ligation reaction with 1 μL vector (P17) and 7 μL water as a transformation control. We transformed 50 μL TOP10 cells with 7 μL DNA and 100 μL DH5α cells with 7 μL DNA. We also used 7 μL of the vector-only ligation to transform 7 μL DH5α cells.
- To test the Lac QPI system, we ligated P45 (RBS+GFP+terminator) to P108. We used a vector to insert ratio of 1:7. We also prepared a ligation reaction with 1 μL vector (P108) and 7 μL water as a transformation control. We transformed 100 μL DH5α cells with 7 μL DNA and we also used 7 μL of the vector-only ligation to transform 7 μL DH5α cells.
