Team:Warsaw/Calendar-Stage1/12 July 2010

From 2010.igem.org

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<br>5.  Ligation of sample 12.1 J23100 [promoter] with the community RBSes-GFP-terminator samples: 7.2 G, 8.1 E, 9.1 A, 11.1 P, 10.1 F
<br>5.  Ligation of sample 12.1 J23100 [promoter] with the community RBSes-GFP-terminator samples: 7.2 G, 8.1 E, 9.1 A, 11.1 P, 10.1 F
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<br><br><div class="note">Cloning fluorescent proteins behind expression vector 3.1 and behind promoter J23100</div>
 
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1. Digest part part 3.1 – Anderson RBS from distribution (expression vector = promoter J23100+RBS)  with SpeI and PstI
 
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<br>2. Digest  fluorescent proteins with XbaI and PstI to clone into an expression vector. Fluorescent proteins:
 
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<br>- GFP E240
 
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<br>- CFP E0420
 
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<br>- YFP E0430
 
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<br>- mOrange E2050*
 
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<br>- mCherry J67102*
 
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<br>- mCherry NLS J130012
 
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<br>* can have an internal RBS. Check by cloning behind J23100.
 
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<br>3. Gel electrophoresis [Gel 4]
 
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<br>4. Gel extraction and ligation of fluorescent proteins with expression vector.
 
<br>______________________________________________
<br>______________________________________________
<br>Electrophoresis (opis zdjęć wg kolejności naniesionych próbek):
<br>Electrophoresis (opis zdjęć wg kolejności naniesionych próbek):
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<br>Gel 2: 7.2, 8.1, 9.1, 10.1 [Connumity RBSes+GFPterminator]
<br>Gel 2: 7.2, 8.1, 9.1, 10.1 [Connumity RBSes+GFPterminator]
<br>Gel 3: 11.1, 12.1 [Community RBS+GFPterminator and J23100]
<br>Gel 3: 11.1, 12.1 [Community RBS+GFPterminator and J23100]
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<br>Gel 4: GFP E240, CFP E0420, YFP E0430, mOrange E2050, mCherry J67102, mCherry NLS J130012
 
<br>______________________________________________
<br>______________________________________________
<br>Digest conditions, used for all reactions:
<br>Digest conditions, used for all reactions:
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<br>RBSes+FPterminator at expected size, vectors at expected size;
<br>RBSes+FPterminator at expected size, vectors at expected size;
<img src="https://static.igem.org/mediawiki/igem.org/d/de/12_07_communityRBS_Xba_Pst.JPG"/>
<img src="https://static.igem.org/mediawiki/igem.org/d/de/12_07_communityRBS_Xba_Pst.JPG"/>
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<div class="note"> GEL 4</div>
 
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Samples: GFP E240, CFP E0420, YFP E0430, mOrange E2050, mCherry J67102, mCherry NLS J130012
 
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<br>Not all samples at expected size, some vectors at wrong size.
 
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<br>Looks like order of some samples is mixed. We will check it **after** transformation, because those are fluorescent proteins therefe it is easy to find the correct samples. Vector with J23100 of expexted size.
 
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<br><img src="https://static.igem.org/mediawiki/igem.org/8/8e/12_07_FluoProteinsXbaPstandVectorSpePst3_1.JPG"/>
 
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Revision as of 20:21, 16 July 2010

Example Tabs

Monday, 12 June 2010; summarised by Milena

Cloning GFP+terminator behind Anderson's RBSes
1. Finish the alkaline lysis from Saturday 10.07, samples: 2.1, 3.1, 4.1, 5.1, 6.1
2. Digest samples 2.1, 3.1, 4.1, 5.1, 6.1 (Anderson's RBSes+GFPterminator) with EcoRI +PstI to examine the correctness of obtained constructs.
3. Gel electrophoresis.[Gel 1]
4. All constructs are of the expected size.

Cloning Connumity RBSes+GFP+terminator behind 23100
1. Digest sample 12.1 J23100[synthetic promoter] with SpeI(Bcu)+ PstI,
2. Gel electrophoresis [Gel 3]
3. Digest community RBSes-GFP-terminator, samples: 7.2 G, 8.1 E, 9.1 A, 11.1 P, 10.1 F with XbaI + PstI
4. Gel electrophoresis [Gel 2, Gel 3]
5. Ligation of sample 12.1 J23100 [promoter] with the community RBSes-GFP-terminator samples: 7.2 G, 8.1 E, 9.1 A, 11.1 P, 10.1 F
______________________________________________
Electrophoresis (opis zdjęć wg kolejności naniesionych próbek):
Gel 1: 2.1, 3.1, 4.1, 5.1, 6.1 [Anderson RBSes+GFPterminator]
Gel 2: 7.2, 8.1, 9.1, 10.1 [Connumity RBSes+GFPterminator]
Gel 3: 11.1, 12.1 [Community RBS+GFPterminator and J23100]
______________________________________________
Digest conditions, used for all reactions:
8 μl DNA
2 μl BamHI buffer
10 μl H2O
0,3 μl enzymów
20 μl final volume
digest for 3h
______________________________________________
Ligation conditions, used for all reactions:
25ul insert DNA
2ul vector DNA
3ul ligase buffer
30 ul final volume
overnight ligation

GEL 2
Samples: 7.2, 8.1, 9.1, 10.1 [Connumity RBSes+GFPterminator]
RBSes+FPterminator at expected size, vectors at expected size;

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