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Team:DTU-Denmark/Notebook/16 August 2013
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Done with primerpair 54 as a two step PCR program with 98C in 10 sec. and 72C for 1:10 min looping 35 times. | Done with primerpair 54 as a two step PCR program with 98C in 10 sec. and 72C for 1:10 min looping 35 times. | ||
Revision as of 12:05, 16 August 2013
16 August 2013
Contents |
lab 208
Main purpose
Who was in the lab
Kristian, Julia, Henrike
Procedure
Gradient PCR for Nir2 USER
Set up new gradient PCR for Nir2 USER to run in 223. Reaction mix without MgCl2 but using DMSO and GC-buffer.
- template: Nir2 extraction fragment
- primers: 40a, 40b
- additives: 5% DMSO
- buffer: GC
Gradient went from 60C to 72C in 12 steps.
| sample # | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| temperature | 60.2 | 60.5 | 61.6 | 63.0 | 64.2 | 65.4 | 66.6 | 67.6 | 68.9 | 70.2 | 71.6 | 72 |
PCR for BioBrick construction
PCR amplified the constructs from Hello World and cytochromes and pSB1C3, which will be ligated together into biobricks.
constructs:
- GC-buffer, 5%DMSO, 2uL 50mM MgCl2
- primers: 53a, 53b
- templates: TAT3-2, Sec2, cycAX
Tried two different annealing temperatures. Program:
| temperature | time | cycles |
|---|---|---|
| 98C | 2:00 | - |
| 98C | 0:10 | 36 |
| 60C/56C | 0:45 | 36 |
| 72C | 0:45 | 36 |
| 72C | 5:00 | - |
| 10C | hold | - |
pSB1C3:
Done with primerpair 54 as a two step PCR program with 98C in 10 sec. and 72C for 1:10 min looping 35 times.
SPL project
Picked single colonies of araBAD SPL transformants and replated them to prepare for Biolector experiment to measure the strength of the expression.
Results
Conclusion
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