Team:DTU-Denmark/Notebook/25 August 2013
From 2013.igem.org
25 August 2013
Navigate to the Previous or the Next Entry
Contents |
Lab 208
Main purpose
Who was in the lab
Procedure
Colony PCR
Performed colony PCR to confirm insert for HAO, AMO, cycAX and Nir transformants. Used Q5 premix with the following reaction mix:
compound | amount |
---|---|
Q5 mix | 25 uL |
FW primer | 3 uL |
RV primer | 3 uL |
template | 1 uL |
MilliQ | 18 uL |
Template was made by resuspending 1 culture in 100uL MilliQ.
program:
temperature | time | cycles |
---|---|---|
98C | 10:00 | - |
98C | 0:10 | 36 |
annealing temperature | 0:30 | 36 |
72C | 0:20 | 36 |
72C | 5:00 | - |
10C | hold | - |
details (primers, temp, expected fragment length):
- cycAX - FW_1, RV_2, 61C, 749bp
- HAO - FW_2, RV_3, 64C, 751bp
- AMO - FW_2, RV_3, 63C, 750bp
- Nir - FW_2, RV_3, 64C, 733bp
- Nir - FW_5, RV_6, 71C, 737bp
Glucose plates
Remade USER ligation and transformation of HAO and cycAX onto plates containing a surplus of glucose. This should reduce the leakiness of the araBAD promoter and improve the health of the transformants before induction so we can recover more colonies.
Results
Conclusion
Navigate to the Previous or the Next Entry