Difference between revisions of "Team:Stony Brook/Notebook/Cancer-W10"
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Revision as of 03:27, 7 September 2016
Week 10: 8/29 - 9/4
8/29
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 |
PCR of Toggle 2
| 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 |
Digests of Toggle parts
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 |
8/30
Ligate Toggle 2 into Plasmid
| Content | ul |
|---|---|
| Ligase Buffer | 2 |
| Toggle pieces | 10 (500 ng/ul) | Plasmid | 2 |
| H2O | 5 |
| Ligase | 1 |
Phosphatase of linearized pSBK3
| Amount | ul |
|---|---|
| Digestion of plasmid | 10 |
| Buffer | 2 | Phosphatase | 1 |
| H2O | 7 |
8/31
Digest of IDT CR-1 SBU and Vector
CR-1 SBU
| Content | Experimental ul | Control ul |
|---|---|---|
| H2O | 18 | 20 |
| Cutsmart | 5.0 | 5.0 |
| EcoRI-HF | 1.0 | X |
| XhoI | 1.0 | X |
| DNA | 25.0 | 25.0 |
pEP352GAP
| Content | Experimental ul | Control ul |
|---|---|---|
| H2O | 7.0 | 8.0 |
| Cutsmart | 1.0 | 1.0 |
| EcoRI-HF | 0.5 | X |
| XhoI | 0.5 | X |
| DNA | 1.0 | 1.0 |
Incubation
- Incubated overnight
9/1
9/2
9/3
9/4
