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Team:DTU-Denmark/Notebook/22 August 2013
From 2013.igem.org
22 August 2013
Contents |
lab 208
Main purpose
Who was in the lab
Kristian, Henrike
Procedure
USER ligation and transformation
Redid for HAO and cyc in arabinose inducible pZA21::araBAD and for Nir fragments
PCR for Biobrick parts
Set up a new PCR reaction for Biobrick parts using HF buffer and 5% DMSO but no MgCl2. PCR was run on a touchdown program
primers: 53a, 53b
template: Sec2 miniprep
program:
| temperature | time | cycles |
|---|---|---|
| 98C | 2:00 | - |
| 98C | 0:10 | 10 |
| 63C | 1:00 | 10 |
| -0.5C per cycle | ||
| 72C | 1:00 | 10 |
| 98C | 0:10 | 25 |
| 53C | 1:00 | 25 |
| 72C | 1:00 | 25 |
| 72C | 5:00 | - |
| 10C | hold | - |
Gel purification
Gel purified the linearized plasmid pZA21::ara with USER endings
colony pPCR to verify AMO insert
Picked 10 colonies from yesterday's cloning for colony PCR. Diluted cells in 50 uL of MilliQ and took 1 uL as template
primers: 53a, 53b
program:
| temperature | time | cycles |
|---|---|---|
| 98C | 10:00 | - |
| 98C | 0:10 | 36 |
| 56C | 0:30 | 36 |
| 72C | 2:00 | 36 |
| 72C | 5:00 | - |
| 10C | hold | - |
Results
nanodrop measurement of ara spl plasmids
Conclusion
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