IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week11/Chemical and Light
Retransformations 9/15
P126 and 127 retransformed into DH5α. Split 30μL and rest onto 2 diff CM plates.
9/16: Retransform went fine, although still fewer cells/DNA could have been used. Colonies picked for overnight cultures.
Ligations 9/15
- Tried the Takara ligation kit: 1μL vector, 4μL insert, 5μL ligation mix
- Transformed into DH5α
mtrB
P63 EX (8/14, dephos) with:
- mtrB TOPO ES (8/15): 0 colonies (on KAN)
- mtrB BB ES (8/14): 3 colonies (on KAN)
P97 SP (dephos) with:
- mtrB BB XP (8/14): TMTC (on AMP)
- mtrB TOPO XP (8/15): TMTC (on AMP)
It seems like there's something wrong with the P97, so if the colony PCRs indicate there's nothing in the plasmids, the tube'll be tossed.
Colony PCR
The 3 mtrB+63 colonies and 4 of each mtrB+97 colonies were picked for colony PCR:
Mix (split into 11): 247.5μL Platinum supermix, 5.5μL BBpfx, 5.5μL BBsfx, 16.5μL H2O
Rx: 5min denature, 35 cycles of (45s denaturation at 94°C, 30s annealing at 55°C, 2m40s at 72°C), 7min final extension, hold at 4°C
2KB ladder used. Clearly, none of the clones actually had a 2kb+ band, so mtrB was not incorporated.
Lac QPI
P3+51 EX (8/3, dephos) with P38 oligo mix (Amy's prep)
0 colonies (on KAN)
RE digests 9/17: mtrB TOPO, 101, 102, 116, 117
The following digests were performed using the Fermentas protocol for 25 min: mtrB TOPO ES; mtrB TOPO XP; P63 EX; P97 SP, P101, 102, 116, 117 ES
Aside from 116, 117, the bands were extracted using a clonewell gel. 116 and 117 were not visible.
P63, P97, and P101 were dephosphorylated according to the NEB instructions using Antarctic phosphatase, and the phosphatase was heat killed.
Ligations 9/18
The following ligations were performed (using 1:6 vector:insert ratio):
- mtrB TOPO ES + P63 EX
- mtrB TOPO XP + P97 SP
- P101 ES + P102 ES
The DNA was mixed, heated to 65°C for 2 minutes, and then mixed with ligase.
- With Takara kit, 6μL DNA mix was added to 6μL ligation mix
- With NEB kit, 9μL DNA mix was added to 1.1μL T4 ligation buffer and 1μL T4 ligase
The mix was cooled to 16°C for 45min and then held at 4°C prior to transformation.
mtrB PCR 9/18
Tried PCR again with primers that incorporate RBS and Platinum Pfx.
| w/ cells | w/ TOPO plasmid | ||||||
|---|---|---|---|---|---|---|---|
| 12x | 12x | ||||||
| Amp buffer | 5 | 60 | Amp buffer | 5 | 60 | ||
| 10mM dNTP | 1.5 | 18 | 10mM dNTP | 1.5 | 18 | ||
| 50 mM MgSO4 | 1 | 12 | 50 mM MgSO4 | 1 | 12 | ||
| Primer F | 0.75 | 9 | Primer F | 0.75 | 9 | ||
| Primer R | 0.75 | 9 | Primer R | 0.75 | 9 | ||
| Pfx | 0.75 | 9 | Pfx | 0.75 | 9 | ||
| Water | 40.25 | 483 | Water | 40 | 480 | ||
| DNA | 0.25 | 3 | |||||
| w/PCR enhancer | |||||||
| Amp buffer | 5 | 60 | Amp buffer | 5 | 60 | ||
| 10mM dNTP | 1.5 | 18 | 10mM dNTP | 1.5 | 18 | ||
| 50 mM MgSO4 | 1 | 12 | 50 mM MgSO4 | 1 | 12 | ||
| Primer F | 0.75 | 9 | Primer F | 0.75 | 9 | ||
| Primer R | 0.75 | 9 | Primer R | 0.75 | 9 | ||
| Pfx | 0.75 | 9 | Pfx | 0.75 | 9 | ||
| PCRx | 5 | 60 | Water | 35 | 420 | ||
| Water | 35.25 | 423 | PCRx | 5 | 60 | ||
| DNA | 0.25 | 3 | |||||
Rx: 2min @ 94°C → 35x[15s @ 94°C → 30s @ {55-61}°C → 2m30s @ 68°C] → 7m @ 68°C → ∞ @ 4°C
