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Team:Freiburg/Notebook/16 August
From 2011.igem.org
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green light receptor
NAME OF YOUR EXPERIMENT
Investigators:NAME
blue light receptor
Picking clones
Investigators: Sandra
Picking clones of transformed cells with LovTAp, NotGate, pSB1T3.
red light receptor
continuing with PCR of cph8
Investigators:Julia
2µl of the PCR reaction from 15th Aug. were loaded onto a gel.
But there was no band. Jakob is going to repeat the PCR.
3a-assembly with promotor+ribosome binding site infront of pcyA and ho1
Investigators:Julia
Digestion
- PR1 in pSB1C3
- PR2 in pSB1C3
- pcyA (pSB1T3)
- pcyA+terminator(d)in pSB1T3
- ho1+Terminator (pSB1T3)
- vector (pSB1K3)
Amount of DNA and H20:
| Sample | 500(ng)/DNA concentration (ng/μl) | H20 μl |
| pcyATd | 2.8 | 35.2 |
| pcyaTb | 2.9 | 35.1 |
| PR2 | 7.2 | 30.8 |
| PR1 | 6.1 | 31.9 |
| S22a | 2,5 | 35.5 |
| pSB1K3 | 20 | 18 |
Enzymes necessary for digestion:
| PR1/PR2 | pcyA/S22a | vector | |
| enzyme 1 | EcoRI | XbaI | EcoRI |
| enzyme 2 | SpeI | PstI | PstI |
-incubated for 1 hours at 37°C.
-heated for 20 minutes at 80°C
Ligation
Ligation of:
| PcyATd | S22a (ho1) | |
| PR1 | 1 pTd | 1 pTb |
| PR2 | 2 pTd | 2 pTb |
PCR
Investigator: Jakob
PCR
| Name:
Jakob | Date:
16.08.2011 |
| Continue from Experiment: 12.08.2011
Order primers | |
| Project Name:
Red light receptor, amplify Cph8 | |
PCR-Mixture for one Reaction:
For a 50 µl reaction use
| 32,5µl | H20 | Name |
| 10µl | 5x Phusion Buffer | of Primer |
| 2.5µl | Primer fw | P 58 (1:10) |
| 2.5µl | Primer dw | P 59 (1:10) |
| 1µl | dNTPs | of Template DNA |
| 1µl | DNA-Template | JT122 5ng/µl |
| 0.5 µl | Phusion (add in the end) |
The program we used:
| | | Nr. of cycles |
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We got the information for the program and the primer sequences from the iGEM Team Uppsala Sweden.
Lysis cassette
2A Assembly like August 15
Investigators:Theo
This time I used all the amount of S15 plasmid DNA we had, did an overnight restriction and run the gel.
extraction as per SOP and ligation with S4 (cut with SpeI, PstI), Transformation in competent cells.
Precipitator
NAME OF YOUR EXPERIMENT
Investigators: NAME
