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	'''DNA-concentration measured with nanodrop:'''<br/>		'''DNA-concentration measured with nanodrop:'''<br/>

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Revision as of 13:44, 27 July 2011

Contents 1 Commons 2 green light receptor 2.1 Testdigest Ligation of CcaS into pSB1K3 3 blue light receptor 3.1 NAME OF YOUR EXPERIMENT 4 red light receptor 4.1 NAME OF YOUR EXPERIMENT 5 Lysis cassette 5.1 NAME OF YOUR EXPERIMENT 6 Precipitator 6.1 NAME OF YOUR EXPERIMENT

Commons

PCR

Name: Sophie	Date: 25.07.11
Continue from Experiment (Date) (Name): Commons
Project Name: more linearized backbones (4 different vectors)

PCR-Mixture for one Reaction:

For a 50 µl reaction use

32,5µl	H20	Name
10µl	5x Phusion Buffer	of Primer
2.5µl	Primer fw	SB-prep-3P-1
2.5µl	Primer dw	SB-prep-2Ea
1µl	dNTPs	of Template DNA
1µl	DNA-Template	PSB 1 A3 PSB 1 C3 PSB 1 K3 PSB 1 T3
0.5 µl	Phusion (add in the end)

What program do you use?

1. 2Min 94°C
2. 30s 94°C
3. 30s 55°C
4. 3min 72°C
5. 10min 72°C

step 2,3 and 4 in 35 cycles

To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.

How did you label the PCR-Product, where is it stored and what do you do next?

Labeled PSB 1 A3, PSB 1 C3, PSB 1 K3 and PSB 1 T3 stored in -20°C in last drawer

green light receptor

Testdigest Ligation of CcaS into pSB1K3

Investigators:JULIA

Testdigest

Continue from Experiment (Date 22.07) Ligation of CcaS into pSB1K3

Project Name: Green light receptor

For one reaction you need For Mastermix: 35 samples+2extra

4μl	H2O	178μl	H2O
1μl	Buffer, NEB4	37μl	Buffer, NEB4
1μl	BSA (10x)	37μl	BSA (10x)
0,5 μl	Enzym 1	7μl	EcoRI
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.

Picture of 1% gel:

result:4a2,4b2,4b3,4b4,4b5 should have the right insert.

blue light receptor

NAME OF YOUR EXPERIMENT

Gibson NOT-Gate

Investigators:

Sophie

Transformation

Name: Sophie	Date: 25.07.11
Continue from Date Name Experiment
Project Name: Blue light (NOT-Gate)

Procedure

1. take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
2. thaw cells on ice 20 minutes
3. pipette 50 μl cells and 2 μl DNA into eppi still on ice!
4. Incubate for 30 minutes on ice
5. Heat at 42°C for 60 sec
6. Incubate on ice for 5 minutes
7. Add 200 μl LB Broth
8. Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
9. Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance

Documentation:

Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.

We need a NOT Gate for our Blue light system. In this experiment I transformed E.coli with the Part Bba_Q04400 from the iGEM distribution kit. The vector is pSBK3 with Kanamycin resistance. The strain ist named S 45 and will be plated out. The plates will be stored in the incubator.

PCR

Name: Sophie	Date: 25.07.11
Continue from Experiment: new experiment. We need NOT-Gate and LovTAP with Gibson-overhangs, so 2 PCRs are made
Project Name: Blue Light

PCR-Mixture for one Reaction:

For a 50 µl reaction use

32,5µl	H20	Name
10µl	5x Phusion Buffer	of Primer:
2.5µl	Primer fw	NOT_G_up LOV_G_up
2.5µl	Primer dw	NOT_G_dw LOV_G_dw
1µl	dNTPs	of Template DNA
1µl	DNA-Template	S35 (BBa_K322999) S45 (BBa_Q04400)
0.5 µl	Phusion (add in the end)

What program do you use?

"57°C auf 70°C" (first annealing temperature:55°c, after 10 cycles 65°c)

To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.

How did you label the PCR-Product, where is it stored and what do you do next?

I labelled it with a heart and a star and stored it in the minipreps 3 box.

I will do Gibson assemblz of the two parts next.

DNA-concentration measured with nanodrop:

sample	DNA-concentration (ng/μl)
S35	75.3
S45	27.2

We will repeat the PCr, because of the bad result of S35. We will do a PCR with higher temperature.

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME

Precipitator

NAME OF YOUR EXPERIMENT

Investigators: NAME