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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
Ligation
| Name: Sophie | Date: 18.08.11 |
| Continue from Date: 18.08.11 Name: Sophie
Experiment: Cloning | |
| Project Name: inducible Promoter for pbd with Cm-vector | |
Procedure
PCR tube:
total volume 20 μl
- add H2O (17 μl -X-Y-Z)
- add 2 μl Ligase Buffer 10x
- add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
- add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
- Add 1 μl T4-DNA Ligase
- Incubate 10-30 min at room temperature
- heat for 20 minutes at 80°C
- store at -20°C or directly proceed to transformation
| Name of part | Ratio Insert:Vector
= 3:1 or 1:1 | Volume (μl) | |
| X insert 1 | S54 | both | 2 or 3 |
| Y insert 2 | PR 444 bzw. PR 668 | 2 or 3 | |
| Z vector | Psb1A3 | 2 or 1 | |
| H2O | 11 |
Documentation:
Why are you doing this experiment? Where are your parts stored? Name the parts for ligation
| Stored in Ligation-box, single parts stored in minipreps, verdaut-box
name of parts see above. |
PCR
Investigators: Rüdiger
| name | templates | primer |
| a | pGex-6-P-1a | P62+P63 |
| b | pGex-6-P-1b | ´´ |
| a10 | pGex-G-P-1a | ´´ |
| b10 | pGex-6-P-1b | ´´ |
PCR did not work. New primer design.
