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Team:Freiburg/Notebook/18 August
From 2011.igem.org
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green light receptor
Mutation PCR of CcaS
Investigators:Jakob
blue light receptor
NAME OF YOUR EXPERIMENT
Investigators:NAME
red light receptor
picked colonies from yesterdays trnasformation.
new PCR in order to again cph8 from pJT122.
last time PCR did not work.
Lysis cassette
NAME OF YOUR EXPERIMENT
Investigators:NAME
Precipitator
Miniprep
zymo Kit
| Name:Sophie
| Date:18.08.11 |
| Continue from Experiment: Cloning: ε5 was empty and the products from the cloning of 12.08.11 contain GFP-pbd too. (Date): 12.08.11
(Name): Sophie | |
| Project Name: inducible Promoter with pbd with cm-vector | |
Documentation:
Why are you doing this experiment? Name the parts which you extract.
ε5 was empty and the products from the cloning of 12.08.11 contain GFP-pbd too.
Name of the parts: GFP-pbd 4 4 4, GFP-pbd 4 1 3, GFP-pbd 6 6 8, GFP-pbd 4 2 2
Describe your results and mistakes and measure the DNA concentration with the Nanodrop and note the results.
| GFP-pbd 4 4 4: 164,1 ng/µl
GFP-pbd 4 1 3: 100,8 ng/µl GFP-pbd 6 6 8: 163,5 ng/µl GFP-pbd 4 2 2: 144,4 ng/µl |
How did you label your probes and where are they stored?
| Labelled GFP-pbd 4 4 4, GFP-pbd 4 1 3, GFP-pbd 6 6 8, GFP-pbd 4 2 2
stored in -20 until sequencing |
Testdigest
| Name: Sophie
| Date: 18.08.11 |
| Continue from Experiment: Miniprep (Date): 18.08.11
(Name): Sophie | |
| Project Name:inducible promoter for pbd with cm-vector | |
For one reaction you need: For Mastermix: Number of samples+2extra
| 4μl | H2O | 20 | |
| 1μl | Buffer, NEB4 | 5 | |
| 1μl | BSA (10x) | 5 | |
| 0,5 μl | Enzym 1 | 2,5 | |
| 0,5 μl | Enzym 2 | 2,5 | |
| 3 μl | DNA |
10 μl total volume
Give 3 μl of DNA in an eppi and add 7μl of the mastermix.
Incubate for about 1h at 37°C.
Add 1 μl Loading dye buffer and load the gel.
Take a picture of the gel, print picture and label the lanes!
3A-assembly of IPTG-promoter, pbd-GFP and Cm-vector
Investigators: Sophie
Digestion
Amount of DNA and H20:
| Sample | DNA μl | H20 μl |
| IPTG-Promoter | 2,5 | 33 |
| GFP-pbd | 3 | 36 |
| pSB1A3 | 20 | 18 |
Enzymes necessary for digestion:
| GFP-pbd: 4 4 4 or 6 6 8 | IPTG-Promoter: S54 | vector: psB1C3 | |
| enzyme 1 | EcoRI | XbaI | EcoRI |
| enzyme 2 | NheI | PstI | PstI |
- Incubation at 37°C for 6 hours
- 20 minutes at 80°C
I won't continue with the IPTG-inducible-promoter-experiments because the part is shit.
We will work with a new IPTG-promoter
