Team:Freiburg/Notebook/4 August

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Contents 1 green light receptor 1.1 Digest with Dpn1 1.2 Transformation 1.3 Testdigest of mutated CcaS 2 blue light receptor 2.1 NAME OF YOUR EXPERIMENT 3 red light receptor 3.1 Testdigest of pcyA and terminator 4 Lysis cassette 4.1 Transformation

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: α β γ δ ε ζ with Kanamycin resistance in pSB1K3 Lys + RBS with Chloramphenicol resistance pSB1C3 Lys complete with tetracycline resistance pSB1T3 Stored: orange rack in the fridge