Team:Warsaw/Calendar-Stage1/12 July 2010

From 2010.igem.org

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Monday, 12 June 2010
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Monday, 12 June 2010; summarised by Milena
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<div class="note">Cloning GFP+terminator behind Anderson's RBSes </div>
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<br><br><div class="note">Cloning GFP+terminator behind Anderson's RBSes </div>
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<br>1. Finish the alkaline lysis from Saturday 10.07, samples: 2.1, 3.1, 4.1, 5.1, 6.1
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<div class="note">Cloning Connumity RBSes+GFP+terminator behind 23100 </div>
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1. Finish the alkaline lysis from Saturday 10.07, samples: 2.1, 3.1, 4.1, 5.1, 6.1
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<br>2. Digest sample 12.1 J23100[synthetic promoter] with SpeI(Bcu)+ PstI,
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<br>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.  
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<br>Gel electrophoresis [Fig X]
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<br>3. Gel electrophoresis.[Gel 1]
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<br>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
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<br>4. All constructs are of the expected size.
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<br>Gel electrophoresis {Fig X]
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<br> 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>
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<br>4. Digest 2.1, 3.1, 4.1, 5.1, 6.1 (RBS Andersona + GFP) [Eco +Pst] w celu sprawdzenia poprawności konstruktów. Elektroforeza. <br>Wszystkie klony są poprawne ( potrzebne zdjęcie żelu!)
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Elektroforeza – zdjęcie (opis zdjęć wg kolejności naniesionych próbek):
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1-szy  żel: 2.1, 3.1, 4.1, 5.1, 6.1
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2-gi żel: 7.2, 8.1, 9.1, 10.1
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3-ci żel: 11.1, 12.1
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<div class="note">Cloning fluorescent proteins behind expression vector 3.1 and behind promoter J23100</div>
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5. Digest part part 3.1 – Anderson RBS from distribution (expression vector = promoter J23100+RBS)  with SpeI and PstI
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6. Digest  fluorescent proteins with XbaI and PstI, klonujemy pod wektor ekspresyjny. Białka fluorescencyjne:
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- GFP E240
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- CFP E0420
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- YFP E0430
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- mOrange E2050* / klonujemy bez RBS-u
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- mCherry J67102*/ klonujemy bez RBS-u
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- mCherry NLS J130012
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* może zawierac internal RBS. Sprawdzic – podklonowac pod promotor.
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Gel – out tych bricków, ligacja z wektorem ekspresyjnym i transformacja
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<br><br><div class="note">Cloning Community RBSes+GFP+terminator behind J23100 </div>
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1. Digest sample 12.1 J23100[synthetic promoter] with SpeI(Bcu)+ PstI,
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<br>2. Gel electrophoresis [Gel 3]
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<br>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
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<br>4. Gel electrophoresis [Gel 2, Gel 3]
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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
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<br>______________________________________________
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<br>Electrophoresis (opis zdjęć wg kolejności naniesionych próbek):
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<br>Gel 1: 2.1, 3.1, 4.1, 5.1, 6.1 [Anderson RBSes+GFPterminator]
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<br>Gel 2: 7.2, 8.1, 9.1, 10.1 [Connumity RBSes+GFPterminator]
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<br>Gel 3: 11.1, 12.1 [Community RBS+GFPterminator and J23100]
<br>______________________________________________
<br>______________________________________________
<br>Digest conditions, used for all reactions:
<br>Digest conditions, used for all reactions:
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<br>20 μl final volume
<br>20 μl final volume
<br>digest for 3h
<br>digest for 3h
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<br>
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<br>______________________________________________
<br>Ligation conditions, used for all reactions:
<br>Ligation conditions, used for all reactions:
<br>25ul insert DNA
<br>25ul insert DNA
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<br>30 ul final volume
<br>30 ul final volume
<br>overnight ligation
<br>overnight ligation
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<div class="note"> GEL 2</div>
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Samples: 7.2, 8.1, 9.1, 10.1 [Community RBSes+GFPterminator]
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<br>RBSes+GFPterminator 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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<br><div class="note">blablabla temat2</div>
 
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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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Latest revision as of 16:35, 31 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 Community RBSes+GFP+terminator behind J23100
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
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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]
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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
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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 [Community RBSes+GFPterminator]
RBSes+GFPterminator at expected size, vectors at expected size;

Retrieved from "http://2010.igem.org/Team:Warsaw/Calendar-Stage1/12_July_2010"