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 Quickchange-modified Lysis Repressor Part (modified K098995)
Right-click to download the annotated ape (.gb) file
3A Assembly (K098995 + K124017)
Digestion of 3A Assembly
| Name: Theo
| Date: 26.07.2011
|
| Continue from Experiment : Quickchange V.3
|
| Project Name: Lysis Cassette V.2 (3A assembly of quickchange-modified K098995 with Lysis genes K124017)
|
Procedure
- add H2O (38μl-DNA )
- 5 μl NEB4 buffer (stored at iGEM’s, -20°C)
- 5 μl 10x BSA (used 1:10 diluted sample stored at iGEM’s, -20°C)
- DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
- 1 μl restriction enzymes (stored at iGEM’s, -20°C)
- heat for 1-2 hours 37°C (6 hours if time)
- heat for 20 minutes 80°C (inactivation of enzymes)
- keep at 4°C if you cannot continue
Restriction enzymes you need to cut the vector, insert1 and insert 2:
| Components
| Vector (μl)
| Insert1 -K098995- and 2 - K124017-(μl)
|
| DNA (500ng)
| 10
| 5
| 7
|
| BSA (10x) (5μl)
|
|
|
|
| NEB4 Buffer (5μl)
|
|
|
|
| Enzyme 1 (1μl)
| EcoRI + DpnI
| EcoRI
| XbaI
|
| Enzyme 2 (1μl)
| PstI
| SpeI
| PstI
|
| H2O (38 μl- DNA)
| 27
| 33
| 31
|
| In total 50 μl
|
|
|
|
Documentation:
Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.
| 3A assembly of quickchange-modified K098995 with Lysis genes K124017.
Quickchange-modified K098995: 132,8ng/µl (Amp)
Lysis genes K124017: (Amp+Kan)
*Parts were run on the gel and verified that the inserts were cut, image accidentaly not saved*
Vector: pSB1T3 + DpnI Digestion
Stored in Theos Box
|
Ligation
| Name: Theo
| Date: 26.07.2011
|
| Continue from: 26.07.2011 Lysis Cassette V.2 Digestion
|
| Project Name: Lysis Cassette V.2 (3A assembly of quickchange-modified K098995 with Lysis genes K124017)
|
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
|
| Name of part
| Ratio Insert:Vector
= 3:1 or 1:1
| Volume (μl)
|
| X insert 1
| Modif. K098995
| 3:1 (1,5*3)
| 4,5
|
| Y insert 2
| K124017
| 3:1 (2*3)
| 6
|
| Z vector
| pSB1T3
| 1:3 (1)
| 1
|
| H2O
|
|
| 5,5
|
Documentation:
Why are you doing this experiment? Where are your parts stored? Name the parts for ligation etc.
| Ligation step of 3A assembly.
How to calculate ratios --> e.g. for K124017
Length of pSB1T3= ca 2200bp
Length of K124017+Vector= ca 4500bp
4500/2200= ca 2
So 2*3 µl (since 3:1 is needed) = 6 µl
|
Transformation
| Name: Theo
| Date:
|
| Continue from : 26.07.2011 Lysis Cassette V.2 Ligation
|
| Project Name: Lysis Cassette V.2 (3A assembly of quickchange-modified K098995 with Lysis genes K124017)
|
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.
| New Composite Part in pSB1T3 Vector with quickchange-modified K098995 and Lysis genes K124017
|
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