IGEM:Groningen/Notebook/iGEM 2011/2011/08/17: Difference between revisions

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==Entry title==
==17-8-11==
* Insert content here...
<br>
<br> Plasmid prep of taRNA-pSB1A3-DT and RBS-GFP-DT (again)
<br> DNA concentrations were around 30ng/μl. De taRNA-pSB1A3-DT were send for sequencing and glycerol stocks were made.
<br> Plasmidpreps of RBS-GFP-DT and taRNA-pSB1A3-DT (1 and 4) were used for vector
<br> Checked sequencing results PBAD/araC-pSB1C3, were alright!:)
<br>
<br> Digestion:
<br> RBS-GFP-DT
<br> 5μl vector
<br> 1μl EcoRI
<br> 1μl XbaI
<br> 3μl Fast digest buffer
<br> 1μl FastAP
<br> 19μl MQ
<br>
<br> taRNAc1-pSB1A3-DT
<br> 7.5μl vector
<br> 1μl EcoRI
<br> 1μl XbaI
<br> 3μl Fast digest buffer
<br> 1μl FastAP
<br> 16.5μl MQ
<br>
<br> taRNAc1-pSB1A3-DT
<br> 5μl vector
<br> 1μl EcoRI
<br> 1μl XbaI
<br> 3μl Fast digest buffer
<br> 1μl FastAP
<br> 19μl MQ
<br>
<br> PBAD
<br> 5μl insert
<br> 1μl EcoRI
<br> 1μl SpeI
<br> 2μl Fast digest buffer
<br> 11μl MQ
<br>
<br> PhybB
<br> 4μl insert
<br> 1μl EcoRI
<br> 1μl SpeI
<br> 2μl Fast digest buffer
<br> 12μl MQ
<br>
<br> Incubate for 1 hour at 37 degrees
<br> After incubation: DNA clean up with PCR purification kit of Roche
<br>
<br> Ligation:
<br> PBAD/araC-RBS-GFP-DT
<br> 8.5 μl vector
<br> 5.7 μl insert
<br> 1μl T4 DNA ligase
<br> 2μl T4 DNA ligase buffer
<br> 2.8μl MQ
<br>
<br> PhybB-taRNA-pSB1A3-DT
<br> 8.5 μl vector
<br> 6.5 μl insert
<br> 1μl T4 DNA ligase
<br> 2μl T4 DNA ligase buffer
<br> 2μl MQ
<br>
<br> Self ligation control:
<br> 8.5 μl vector
<br> 1μl T4 DNA ligase
<br> 2μl T4 DNA ligase buffer
<br> 8.5μl MQ
<br>
<br> incubate the samples for 40 minutes at room temperature
<br> For transformation: take a sample of the plasmid PhybB-RBS-GFP-DT (5μl of the plasmid) and transform this in TOP10 competent <br> cells in parallel with the other samples. This will be done because of the DH5alpha strain does not grow well in M9 medium for
<br> flow cytometry measurements...
<br>
<br> Transformation:
<br> Add to 40μl competent cells 10μl ligation mixture
<br> Incubate for 30 min on ice
<br> Heat shock: 45s at 42 degrees
<br> Incubate cells on ice for 2 min
<br> Add 1ml LB medium+ 25mM glucose
<br> Incubate cells for 1h/1.5h at 37 degrees
<br> Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates
<br> Put the plates in the stove at 37 degrees overnight
<br>
<br> Also, in the morning, the plate with PhybB-RBS-GFP looked alright, had single colonies, so I let them grow this morning in 30 <br> degrees for induction of the PhybB promotor. After 1 and 3 hours, no fluorescent colonies were seen on the plate.
 
 




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17-8-11



Plasmid prep of taRNA-pSB1A3-DT and RBS-GFP-DT (again)
DNA concentrations were around 30ng/μl. De taRNA-pSB1A3-DT were send for sequencing and glycerol stocks were made.
Plasmidpreps of RBS-GFP-DT and taRNA-pSB1A3-DT (1 and 4) were used for vector
Checked sequencing results PBAD/araC-pSB1C3, were alright!:)

Digestion:
RBS-GFP-DT
5μl vector
1μl EcoRI
1μl XbaI
3μl Fast digest buffer
1μl FastAP
19μl MQ

taRNAc1-pSB1A3-DT
7.5μl vector
1μl EcoRI
1μl XbaI
3μl Fast digest buffer
1μl FastAP
16.5μl MQ

taRNAc1-pSB1A3-DT
5μl vector
1μl EcoRI
1μl XbaI
3μl Fast digest buffer
1μl FastAP
19μl MQ

PBAD
5μl insert
1μl EcoRI
1μl SpeI
2μl Fast digest buffer
11μl MQ

PhybB
4μl insert
1μl EcoRI
1μl SpeI
2μl Fast digest buffer
12μl MQ

Incubate for 1 hour at 37 degrees
After incubation: DNA clean up with PCR purification kit of Roche

Ligation:
PBAD/araC-RBS-GFP-DT
8.5 μl vector
5.7 μl insert
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
2.8μl MQ

PhybB-taRNA-pSB1A3-DT
8.5 μl vector
6.5 μl insert
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
2μl MQ

Self ligation control:
8.5 μl vector
1μl T4 DNA ligase
2μl T4 DNA ligase buffer
8.5μl MQ

incubate the samples for 40 minutes at room temperature
For transformation: take a sample of the plasmid PhybB-RBS-GFP-DT (5μl of the plasmid) and transform this in TOP10 competent
cells in parallel with the other samples. This will be done because of the DH5alpha strain does not grow well in M9 medium for
flow cytometry measurements...

Transformation:
Add to 40μl competent cells 10μl ligation mixture
Incubate for 30 min on ice
Heat shock: 45s at 42 degrees
Incubate cells on ice for 2 min
Add 1ml LB medium+ 25mM glucose
Incubate cells for 1h/1.5h at 37 degrees
Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates
Put the plates in the stove at 37 degrees overnight

Also, in the morning, the plate with PhybB-RBS-GFP looked alright, had single colonies, so I let them grow this morning in 30
degrees for induction of the PhybB promotor. After 1 and 3 hours, no fluorescent colonies were seen on the plate.



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