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Team:DTU-Denmark/Notebook/3 September 2013
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3 September 2013
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Lab 208
Main purpose
- Biobrick construction -> CycAX and AMO plasmid isolation.
Who was in the lab
Kristian, Julia
Procedure
Midiprep CycAX col.2, AMO col.2
We want a high yield purification. 100mL of ON cultures were pelleted. The protocol provided by QIAGEN Plasmid Kit was followed.
Colony PCR to confirm HAO insert
Used Q5 master mix and made only 25uL reaction volume because there was not enough Q5 mix left to make 50uL.
| compound | volume (uL) |
|---|---|
| Q5 | 12,5 |
| FW | 1,5 |
| RV | 1,5 |
| template | 1 |
| MQ | 8,5 |
Made doubles for two different primer pairs:
- FW_1_HAO_Seq, RF_2_HAO_Seq - 61C - expected fragment length: 742bp
- FW_3_HAO_Seq, RV_3_HAO_Seq - 65C - expected fragment length: 743bp
Program:
| temperature | time | cycles |
|---|---|---|
| 98C | 10:00 | - |
| 98C | 0:10 | 36 |
| annealing temperature | 0:30 | 36 |
| 72C | 0:30 | 36 |
| 72C | 5:00 | - |
| 10C | hold | - |
Results
Conclusion
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