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
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
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| Continue from Date: 30.08.11 Name: Sophie
Experiment: new
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| Project Name:inducible promoter for pbd (iGEM standard)
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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)
- add 5,6 μl antarctic phosphatase buffer and 1 μl antarctic phosphatase and incubate for another hour
- heat for 20 minutes 80°C (inactivation of enzymes)
- 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)
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| GFP-pbd
| 164
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| IPTG-promoter ε 5
| 217
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| psB1C3
| 58
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Restriction enzymes you need to cut the vector, insert1 and insert 2:
| Components
| Vector (μl)
| Insert1 and 2 (μl)
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| DNA (500ng)
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| BSA (10x) (5μl)
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| NEB4 Buffer (5μl)
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| Enzyme 1 (1μl)
| Eco
| Eco
| Xba
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| Enzyme 2 (1μl)
| Pst
| Spe
| Pst
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| H2O (38 μl- DNA)
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| In total 50 μl
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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
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