Team:Stony Brook/Notebook/Cancer-W10
Week 10: 8/29 - 9/4
AmilCP BioBrick Digests
| Content |
Experimental ul |
Control ul |
| H2O |
46.5 |
47.0 |
| Cutsmart |
5.0 |
5.0 |
| XbaI |
0.5 |
X |
| DNA |
2.0 |
2.0 |
PCR of Toggle 1
| Contents |
ul |
Phire |
10 |
| H2O |
8 |
| 5 uM F Primer |
1.5 |
| 5 uM R Primer |
1.5 |
| 100pg/ul Construct |
1.0 |
- Ran other trials with 2.5, 3.5, 4.5 and 6.5 ul of Forward and Reverse Primers
- Water ul was adjusted to compensate: 6, 4, 2 and 0 ul respectively
| Phase |
Temperature (°C) |
Time (sec) |
| Initial Denaturation |
98 |
30 |
| Denaturation |
98 |
5 |
| Annealing |
68 |
5 |
| Extension |
72 |
45 |
| Final Extension |
72 |
60 |
| Contents |
ul |
Phire |
10 |
| H2O |
8 |
| 5 uM F Primer |
1.5 |
| 5 uM R Primer |
1.5 |
| 100pg/ul Construct |
1.0 |
- Ran other trials with 2.5, 3.5, 4.5 and 6.5 ul of Forward and Reverse Primers
- Water ul was adjusted to compensate: 6, 4, 2 and 0 ul respectively
| Phase |
Temperature (°C) |
Time (sec) |
| Initial Denaturation |
98 |
30 |
| Denaturation |
98 |
5 |
| Annealing |
68 |
5 |
| Extension |
72 |
45 |
| Final Extension |
72 |
60 |
Toggle 1 Digest
| Content |
Experimental ul |
Control ul |
| H2O |
24 |
25 |
| Cutsmart |
5.0 |
5.0 |
| SpeI |
1.0 |
X |
| DNA |
25.0 |
25.0 |
Toggle 1 Digest
| Content |
Experimental ul |
Control ul |
| H2O |
24 |
25 |
| Cutsmart |
5.0 |
5.0 |
| XbaI |
1.0 |
X |
| DNA |
25.0 |
25.0 |
Linearized pSB1C7 Digest
| Content |
Experimental ul |
Control ul |
| H2O |
16.5 |
18.5 |
| Cutsmart |
2.5 |
2.5 |
| SpeI-HF |
1.0 |
X |
| XbaI |
1.0 |
X |
| DNA |
4.0 |
4.0 |
Ligation of Toggle parts
| Content |
ul |
| 10X T4 Buffer |
2.0 |
| Toggle 1 |
5.0 |
| Toggle 2 |
10.0 |
| H2O |
2.0 |
| Ligase |
1.0 |
Ligate Toggle 2 into Plasmid
| Content |
ul |
| 10X T4 Buffer |
|
| Vector |
# (# ng) |
Construct |
# (# ng) |
| H2O |
# |
| Ligase |
# |
