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Team:Freiburg/Notebook/18 August
From 2011.igem.org
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green light receptor
Quickchange
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
2A assembly
Investigators:Theo
Sent for sequencing
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.
