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Team:Freiburg/Notebook/4 August
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==<span style="color:green;">green light receptor</span>== | ==<span style="color:green;">green light receptor</span>== | ||
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2µl Dpn1 <br/> | 2µl Dpn1 <br/> | ||
38µl DNA from quickchange (CcaR) <br/> | 38µl DNA from quickchange (CcaR) <br/> | ||
| - | incubate at | + | incubate at 37°C for 1,5 h <br/> |
inactivate at 80°C for 20 min. <br/> | inactivate at 80°C for 20 min. <br/> | ||
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'''Investigator: Jakob''' | '''Investigator: Jakob''' | ||
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'''Documentation:''' | '''Documentation:''' | ||
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* (qc) CcaR in pSB1C3 (Cm resistance) | * (qc) CcaR in pSB1C3 (Cm resistance) | ||
* Stored in the fridge | * Stored in the fridge | ||
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*To-do: pick some colonies | *To-do: pick some colonies | ||
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===Testdigest of mutated CcaS=== | ===Testdigest of mutated CcaS=== | ||
'''Investigator: Julia''' | '''Investigator: Julia''' | ||
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'''Investigators: Jakob''' | '''Investigators: Jakob''' | ||
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name samples: | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name samples: | ||
| - | α β γ δ ε ζ with Kanamycin resistance in pSB1K3 | + | * α β γ δ ε ζ with Kanamycin resistance in pSB1K3 |
| - | Lys + RBS with Chloramphenicol resistance pSB1C3 | + | * Lys + RBS with Chloramphenicol resistance pSB1C3 |
| - | Lys complete with tetracycline resistance pSB1T3 | + | * Lys complete with tetracycline resistance pSB1T3 |
| - | Stored: orange rack in the fridge | + | * Stored: orange rack in the fridge |
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Latest revision as of 01:05, 22 September 2011
Contact 
Contents |
green light receptor
Digest with Dpn1
Investigator: Jakob
5µl Buffer NEB4
5µl BSA (10x)
2µl Dpn1
38µl DNA from quickchange (CcaR)
incubate at 37°C for 1,5 h
inactivate at 80°C for 20 min.
Transformation
Investigator: Jakob
| Name:
Jakob | Date:
04.08.2011 |
| Continue from Experiment: 03.08.2011
Quickchange | |
| Project Name: Green light receptor CcaR | |
Documentation:
- (qc) CcaR in pSB1C3 (Cm resistance)
- Stored in the fridge
- To-do: pick some colonies
Testdigest of mutated CcaS
Investigator: Julia
done a miniprep of the quickchanged CcaS-colonies.
The one CcaS (labeled qC1) was changed with Primer P14 and 15, in order to get rid of EcoRI.
It was digested with EcoRI.
The other CcaS with Primer 16 and 17, was performed to delete SpeI site.
The transformation gave more positive colonies so we have 11 samples.
They were digested with SpeI
For Mastermix: 10+2extra
| 4μl | H2O | 48 | H2O |
| 1μl | Buffer, NEB4 | 12 | Buffer, NEB4 |
| 1μl | BSA (10x) | 12 | BSA (10x) |
| 0,5 μl | Enzym 1 | 1.5 | SpeI |
| 0,5 μl | Enzym 2 | - | |
| 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.
Gelpicture: see red light receptor, lower lane.
Samples C3e and C3c were send to GATC for sequencing.
blue light receptor
NAME OF YOUR EXPERIMENT
Investigators:NAME
red light receptor
Testdigest of pcyA and terminator
Investigators:Julia
For Mastermix: 7+2extra
| 4μl | H2O | 36 | H2O |
| 1μl | Buffer, NEB4 | 9 | Buffer, NEB4 |
| 1μl | BSA (10x) | 9 | BSA (10x) |
| 0,5 μl | Enzym 1 | 1 | EcoRI |
| 0,5 μl | Enzym 2 | 1 | PstI |
| 3 μl | DNA |
10 μl total volume
Give 3 μl of DNA in an eppi and add 7μl of the mastermix.
upper lane: (from left to right)Ladder 1kb, pcyA+terminator, a,b,d,e,f,g,h, last CcaS1 digested with EcoRI,Ladder 1kb
lower lane:Ladder 1kb,all qCcaS, from testdigest, see Greenlight recptor
samples named( from left to right) C3e,C2d,C3b,C3c,C3d,C3h,C2e,C2f,C2g
Lysis cassette
Transformation
Investigators: Jakob
| Name:
Jakob | Date:
04.08.2011 |
| Continue from Experiment: 03.08.2011
Quickchange (Theo), 3A assembly | |
| Project Name: Lysis casette | |
Documentation:
Name samples:
|
