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
Cloning scheduleDate: 30.08.
Name: Rüdiger
1,2,3 : digest: Dpn1+BamH1+EcoR1
>PCR purification
>ligate into David Vector
8:digest:Dpn19EcoR1+psT1
>PCR purification
>ligate into CM Vector (Julia 25.08.)
4+9:PCR purification
>Gibson-Assembly (see SoP)
>PCR with P47+P44/45/46
>digest with Dpn1+EcoR1+PsT1
>ligate into PET-DUET1 Vector
>digest with Dpn1+EcoR1+Spe1
>ligate into CM vector
All PCR at 37°c for 2 hours without buffer.
Add all vectors to digest with Antarctic Phosphatase + AP buffer (5x).
| Name
Rüdiger
| Date:
30.08.2011
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| Continue from Experiment PCR (Date) 29.08.
(Name) Rüdiger
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| Project Name: Blue light receptor: Ligation of Lovtap and Not-Gate
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Gibson-Assembly
1. Prepare 5X ISO buffer. Six ml of this buffer can be prepared by combining the following:
3 ml of 1 M Tris-HCl pH 7.5150 μl of 2 M MgCl260 μl of 100 mM dGTP 60 μl of 100 mM dATP 60 μl of 100 mM dTTP60 μl of 100 mM dCTP300 μl of 1 M DTT 1.5 g PEG-8000300 μl of 100 mM NADAdd water to 6 ml Aliquot 100 μl and store at -20 °C
2. Prepare an assembly master mixture. This can be prepared by combining the following:
320 μl 5X ISO buffer0.64 μl of 10 U/ μl T5 exo20 μl of 2 U/μl Phusion pol160 μl of 40 U/μl Taq ligAdd water to 1.2 ml
Aliquot 15 μl and store at -20 °C. This assembly mixture can be stored at -20 °C for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles. This is ideal for the assembly of DNA molecules with 20-150 bp overlaps. For DNA molecules overlapping by larger than 150 bp, prepare the assembly mixture by using 3.2 μl of 10 U/ μl T5 exo.
3. Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.
4. Add 5 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).
5. Incubate at 50 °C for 15 to 60 min (60 min is optimal).
6. If cloning is desired, electroporate 1 μl of the assembly reaction into 30 μl electrocompetent E. coli.
Documentation:
Why are you doing this experiment? Name the parts for the Gibson-Assembly.
| Precipitator 2 : 4+9
42: 5+10
4:6+11
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Describe your results and mistakes. Did you digest it? Results?
| See gel next day
>Did not work due to wrong DNA concentrations and no PCR purification
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How did you label your samples and where are they stored?
Digestion this time the vector is dephosphorilated by antarctic phosphatase to avoid religation.
| Name: Sophie
| Date: 31.08.11
|
| 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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