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From 2011.igem.org

Contents 1 green light receptor 1.1 NAME OF YOUR EXPERIMENT 2 blue light receptor 2.1 NAME OF YOUR EXPERIMENT 3 red light receptor 3.1 NAME OF YOUR EXPERIMENT 4 Lysis cassette 4.1 NAME OF YOUR EXPERIMENT 5 Precipitator

green light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

blue light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME

Precipitator

Ligation

Name: Ruediger	Date: 20.07
Continue from Experiment Digestion Date 19.07 Name Ruediger
Project Name: GFP Pbd

Procedure

PCR tube:

total volume 20 μl

1. add H₂O (17 μl -X-Y-Z)
2. add 2 μl Ligase Buffer 10x
3. add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
4. add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
5. Add 1 μl T4-DNA Ligase
6. Incubate 10-30 min at room temperature
7. heat for 20 minutes at 80°C
8. 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	--	--	--
Y insert 2	M14+P28+P18/19/20	2.7:1	2 each
Z vector	S39 / S40		2 each
H2O

Documentation:

Why are you doing this experiment? Where are your parts stored? Name the parts for ligation etc.

Ligation of 3 different GFP Pbd part (M14+P28+P18/19/20) into two vector with Promotor and RBS S39 + M14+P28+P18 S39 + M14+P28+P19 S39 + M14+P28+P20 S43 + M14+P28+P18 S43 + M14+P28+P19 S43 + M14+P28+P20

Transformation

Name: Ruediger	Date: 20.07
Continue from Experiment Ligation Date 19.07 Name Ruediger
Project Name: GFP Pbd

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.

1 S39 + M14+P28+P18 2 S39 + M14+P28+P19 3 S39 + M14+P28+P20 4 S43 + M14+P28+P18 5 S43 + M14+P28+P19 6 S43 + M14+P28+P20 All have cm resistance in incubator 37°C overnight