Team:Freiburg/Notebook/30 August

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Contents 1 green light receptor 1.1 Ligation of Promotor PcpcG in PSB1C3 2 blue light receptor 2.1 Transformation 3 red light receptor 3.1 gel extraction 4 Lysis cassette 4.1 NAME OF YOUR EXPERIMENT 5 Precipitator

green light receptor

Ligation of Promotor PcpcG in PSB1C3

Investigators:Julia

1. Digestion of PCR Product from yesterday[1]

38µl PCR product
5µl BSA (10x)
5µl NEB buffer 4
1µl EcoRI
1µlPstI

Incubation for 3 hours, afterwards the sample was cleaned with a PCR purification kit from Qiagen.

2.Ligation

11µl H2O
4µl PcpcG
2µl pSB1C3
2µl T4 Ligase buffer
1µl T4 Ligase

Incubation for 3 hours at room temperature.
heat inactivation at 80 degrees for 20 min.

3. transformation

blue light receptor

Transformation

...with ♥-A3-Not, ♥-A3-nOt and ♥-A3-noT </br>

Investigators: Sophie

red light receptor

gel extraction

Investigators:Julia

Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME

Precipitator

Digestion this time the vector is dephosphorilated by antarctic phosphatase to avoid religation.

Name: Sophie	Date: 31.08.11
Continue from Date: 30.08.11 Name: Sophie Experiment: new
Project Name:inducible promoter for pbd (iGEM standard)

Procedure

1. add H₂O (38μl-DNA )
2. 5 μl NEB4 buffer (stored at iGEM’s, -20°C)
3. 5 μl 10x BSA (used 1:10 diluted sample stored at iGEM’s, -20°C)
4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
5. 1 μl restriction enzymes (stored at iGEM’s, -20°C)
6. heat for 1-2 hours 37°C (6 hours if time)
7. add 5,6 μl antarctic phosphatase buffer and 1 μl antarctic phosphatase and incubate for another hour
8. heat for 20 minutes 80°C (inactivation of enzymes)
9. keep at 4°C if you cannot continue

Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:

Sample Name	DNA concentration (μg/μl)
GFP-pbd	164
IPTG-promoter ε 5	217
psB1C3	58

Restriction enzymes you need to cut the vector, insert1 and insert 2:

Components	Vector (μl)		Insert1 and 2 (μl)
DNA (500ng)
BSA (10x) (5μl)
NEB4 Buffer (5μl)
Enzyme 1 (1μl)		Eco		Eco	Xba
Enzyme 2 (1μl)		Pst		Spe	Pst
H2O (38 μl- DNA)
In total 50 μl

Documentation:

Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.

I want to be able to send the plastic binding domain to the registry and I want it to be expressible. Stored in: “Minipreps, verdaut”-box Resistance: Cm