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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 | - |
Four different reaction mixes: HF, HF+5%DMSO, GC, GC+5%DMSO
pZA21::ara for Nir:
| temperature | time | cycles |
|---|---|---|
| 98C | 2:00 | - |
| 98C | 0:10 | 36 |
| 60C | 1:00 | 36 |
| 72C | 2:00 | 36 |
| 72C | 5:00 | - |
| 10C | hold | - |
Standard reaction mix.
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 | - |
Mix with GC, 5%DMSO and 2uL MgCl2.
pSB1C3 and pZA21::ara for Nir were without results (see gel picture). New PCRs were set up with one standard reaction and one reaction with additives. The annealing temperature was lowered.
second round of PCRs:
| 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 |
pSB1C3:
| temperature | time | cycles |
|---|---|---|
| 98C | 2:00 | - |
| 98C | 0:10 | 36 |
| 60C | 1:00 | 36 |
| 72C | 1:30 | 36 |
| 72C | 5:00 | - |
| 10C | hold | - |
pZA21::ara for Nir:
| temperature | time | cycles |
|---|---|---|
| 98C | 2:00 | - |
| 98C | 0:10 | 36 |
| 55C | 1:00 | 36 |
| 72C | 2:00 | 36 |
| 72C | 5:00 | - |
| 10C | hold | - |
Additionally set up a PCR for cycAX with USER endings since the amount of fragment is running low.
cycAX for USER:
| temperature | time | cycles |
|---|---|---|
| 98C | 2:00 | - |
| 98C | 0:20 | 36 |
| 57C | 0:45 | 36 |
| 72C | 1:30 | 36 |
| 72C | 5:00 | - |
| 10C | hold | - |
Standard reaction mix.
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
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