"
Team:DTU-Denmark/Notebook/27 August 2013
From 2013.igem.org
27 August 2013
Contents |
lab 208
Main purpose
Who was in the lab
Procedure
PCR reactions
Set up PCR reactions for:
- Biobrick backbone pSB1C3 with USER endings fitting for our inserts
- constitutive reference promoter in pZA21 (for SPL)
- pZA21::ara vector with USER endings fitting for Nir1 and Nir2 inserts
first round of PCRs:
pSB1C3:
| temperature | time | cycles |
|---|---|---|
| 98C | 2:00 | - |
| 98C | 0:10 | 36 |
| 70C | 0:45 | 36 |
| 72C | 1:30 | 36 |
| 72C | 5:00 | - |
| 10C | hold | - |
pZA21::ara for Nir:
| temperature | time | cycles |
|---|---|---|
| 98C | 2:00 | - |
| 98C | 0:20 | 36 |
| 60C | 1:00 | 36 |
| 72C | 2:00 | 36 |
| 72C | 5:00 | - |
| 10C | hold | - |
constitutive reference promoter:
| temperature | time | cycles |
|---|---|---|
| 98C | 2:00 | - |
| 98C | 0:20 | 36 |
| 58.1C | 1:00 | 36 |
| 72C | 3:00 | 36 |
| 72C | 5:00 | - |
| 10C | hold | - |
| compound | amount (in uL) | |
|---|---|---|
| standard mix | additive mix | |
| dNTPs | 1 | 1 |
| X7 ploymerase | 0.5 | 0.5 |
| HF buffer | 10 | 10 |
| MilliQ | 31.5 | 27 |
| FW primer | 3 | 3 |
| RV primer | 3 | 3 |
| template | 1 | 1 |
| DMSO | - | 2.5 |
| 50 mM MgCl2 | - | 2 |
Template was made by resuspending 1 culture in 100uL MilliQ.
program:
Results
Gel
- 1 kb ladder
- pSB1C3 for Biobricks, HF buffer
- pSB1C3 for Biobricks, HF buffer, 5%DMSO
- pSB1C3 for Biobricks, GC buffer
- pSB1C3 for Biobricks, GC buffer, 5%DMSO
- negative pSB1C3 for Biobricks
- pZA21::ara for USER ligation with Nir
- pZA21::ara for USER ligation with Nir (duplicate)
- 1 kb ladder
Note: It seems the ladder overflew.
Conclusion
Navigate to the Previous or the Next Entry
