Team:Freiburg/Notebook/26 July

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blue light receptor

Gibson-Assembly

Investigators: Sandra, Sophie

Planned Gibson-Assembly for Lov-tap and Not-Gate. Need to order NAD. We will start an thursday.

Precipitator

Ligation

Name: Ruediger	Date: 26.07
Continue from Date Name Experiment Digestion 25.07 Ruediger
Project Name:GFPPbd

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

Part name	Volume (μl)	Part name	Volume (μl)
S39	2	S43	2
P18	1,8	P18	1,8
Psb1T3	2	Psb1T3	2
S39	2	S43	2
P19	1,8	P19	1,8
Psb1T3	2	Psb1T3	2
S39	2	S43	2
P20	1,8	P20	1,8
Psb1T3	2	Psb1T3	2

Added 12,2 μl to every sample

Documentation:

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

See digestion yesterday 3A assembly combination as shown in table.

Transformation

Name: RT	Date: 26.07
Continue from Date Name Experiment Ligation 26.07 Ruediger
Project Name: GFPPbd

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.

Had only 6 tet plates -> put 100 μl of each sample on each plate Incubator overnight for 30°C S39-P18 S39-P19 S39-P20 S43-P18 S43-P19 S43-P20