IGEM:Groningen/Notebook/iGEM 2011/2011/07/01

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(Autocreate 2011/07/01 Entry for IGEM:Groningen/Notebook/iGEM_2011)
Current revision (07:02, 21 September 2011) (view source)
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==Entry title==
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==1-7-11==
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* Insert content here...
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<br> Calculate with the ligation calculator how much of the insert is needed for a vector: insert ratio of 1:6
 +
<br>
 +
<br> Digestion:
 +
<br> 3μl RBS-GFP-DT
 +
<br> 1μl EcoRI
 +
<br> 1μl XbaI
 +
<br> 2μl FD buffer
 +
<br> 13μl MQ water
 +
<br> insert:
 +
<br> 4μl insert
 +
<br> 1μl EcoRI
 +
<br> 1μl SpeI
 +
<br> 2μl FD buffer
 +
<br> 12μl MQ water
 +
<br>
 +
<br> Digestion one reaction:
 +
<br> 1μl pSB1C3 (50 ng)
 +
<br> 0.5μl insert
 +
<br> 0.5μl EcoRI
 +
<br> 0.5μl PstI
 +
<br> 2μl FD buffer
 +
<br> 15.5μl MQ water
 +
<br>
 +
<br> Digest for 1h at 37 degrees
 +
<br>
 +
<br> DNA clean up with High Pure PCR Purification Kit
 +
<br>
 +
<br> Ligation:
 +
<br> For the 1 digestion reaction: elute in the purification step in 17μl
 +
<br> Add: 2μl T4 DNA ligase buffer and 1μl T4 DNA ligase
 +
<br>
 +
<br> Ligation promotors upstream RBS-GFP-DT
 +
<br> 2μl T4 DNA ligase buffer
 +
<br> 1μl T4 DNA ligase
 +
<br> 8.5μl vector
 +
<br> 6μl insert
 +
<br> 2.5μl MQ water
 +
<br>
 +
<br> Transformation
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<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
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<br> Incubate cells for 1h/1.5h at 37 degrees
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<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
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<br>
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1-7-11


Calculate with the ligation calculator how much of the insert is needed for a vector: insert ratio of 1:6

Digestion:
3μl RBS-GFP-DT
1μl EcoRI
1μl XbaI
2μl FD buffer
13μl MQ water
insert:
4μl insert
1μl EcoRI
1μl SpeI
2μl FD buffer
12μl MQ water

Digestion one reaction:
1μl pSB1C3 (50 ng)
0.5μl insert
0.5μl EcoRI
0.5μl PstI
2μl FD buffer
15.5μl MQ water

Digest for 1h at 37 degrees

DNA clean up with High Pure PCR Purification Kit

Ligation:
For the 1 digestion reaction: elute in the purification step in 17μl
Add: 2μl T4 DNA ligase buffer and 1μl T4 DNA ligase

Ligation promotors upstream RBS-GFP-DT
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
8.5μl vector
6μl insert
2.5μl MQ water

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



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