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Revision as of 02:53, 12 October 2016
Week 14: 9/26 - 10/2
9/26
9/27
9/28
9/29
Toggle 2 Gel Construct using Phire Polymerase
PCR Ran of Toggle 2:
| Phase | Temperature (°C) | Time (sec) |
|---|---|---|
| Initial Denaturation | 98 | 60s |
| Denaturation | 98 | 20s |
| Annealing | Ranged from 72-60 degrees for each tube. Did a different annealing temperature for each PCR. | 10s |
| Extension | 72 | # |
| Final Extension | 72 | # |
Ran 32 cycles
9/30
10/1
Constructs of T1 and T2
- Ran a gel of the constructs
- Set up tubes for TSS Competent cells
CRISBU
| Content | Experimental ul |
|---|---|
| Cutsmart | 5 |
| EcoRI-HF | 1 |
| XhoI | 1 |
| DNA | 43 |
Toggle 1
| Content | Experimental ul |
|---|---|
| Cutsmart | 5 |
| EcoRI-HF | 1 |
| SpeI-HF | 1 |
| DNA | 28 |
- Filled to 50ul with water
Toggle 2
| Content | Experimental ul |
|---|---|
| Cutsmart | 5 |
| EcoRI-HF | 1 |
| PstI-HF | 1 |
| DNA | 35 |
- Filled to 50ul with water
Ligations
pEPGAP
| Content | ul |
|---|---|
| 10X T4 Buffer | 2 |
| Vector | 2 | Insert | 0 |
| H2O | 15 |
| Ligase | 1 |
pEPGAP/CRISBU
| Content | ul |
|---|---|
| 10X T4 Buffer | 2 |
| Vector | 2 | Insert | 2 |
| H2O | 13 |
| Ligase | 1 |
pEPGAP-P
| Content | ul |
|---|---|
| 10X T4 Buffer | 2 |
| Vector | 4 | Insert | 0 |
| H2O | 13 |
| Ligase | 1 |
pEPGAP-P/CRISBU
| Content | ul |
|---|---|
| 10X T4 Buffer | 2 |
| Vector | 4 | Insert | 4 |
| H2O | 8 |
| Ligase | 1 |
pSBIA2/T1/T2
| Content | ul |
|---|---|
| 10X T4 Buffer | 2 |
| Vector | 1 | Insert | 13.52 |
| H2O | 2.5 |
| Ligase | 1 |
pSBIA2
| Content | ul |
|---|---|
| 10X T4 Buffer | 2 |
| Vector | 1 | Insert | 0 |
| H2O | 16 |
| Ligase | 1 |
pSBIA2-p
| Content | ul |
|---|---|
| 10X T4 Buffer | 2 |
| Vector | 2 | Insert | 0 |
| H2O | 15 |
| Ligase | 1 |
pSBIA2-P/T1/T2
| Content | ul |
|---|---|
| 10X T4 Buffer | 2 |
| Vector | 2 | Insert | 13.50 |
| H2O | 2.5 |
| Ligase | 1 |
10/2
Transformations
- Thaw TSS cells on ice
- Add 2ul of prepped plasmid. Pipette to mix
- Sit for 30 minutes on ice
- Incubate cells for 30s at 43°C
- Incubate on ice for 2 minutes
- Add 1ml of 50C
- Incubate for 30 minutes at 37°C. Shake
- Spread 100-300ul on plates
- Grow overnight at 37°C
- Save the rest as liquid cultures
Plates Used:
- Plate 1: pEPGAP
- Plate 2: pEPGAP/CRISBU
- Plate 3: pEPGAP-P
- Plate 4:
- Plate 5: pSBIA2/T1/T2
- Plate 6: pSBIA2
- Plate 7: pSBIA2-P
- Plate 8:
