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
- add H2O (17 μl -X-Y-Z)
- add 2 μl Ligase Buffer 10x
- add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
- add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
- Add 1 μl T4-DNA Ligase
- Incubate 10-30 min at room temperature
- heat for 20 minutes at 80°C
- 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
- take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
- thaw cells on ice 20 minutes
- pipette 50 μl cells and 2 μl DNA into eppi still on ice!
- Incubate for 30 minutes on ice
- Heat at 42°C for 60 sec
- Incubate on ice for 5 minutes
- Add 200 μl LB Broth
- Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
- 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
|
Lysis cassette
Sequencing of Quickchanged Lysis Repressor Part (modified K098995)
https://static.igem.org/mediawiki/2011/3/36/Freiburg11_seq_qcLysepromotor_2607.gb
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