Team:Warsaw/Calendar-Stage1/12 July 2010

From 2010.igem.org

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<div class="note">Cloning GFP+terminator behind Anderson's RBSes </div>
<div class="note">Cloning GFP+terminator behind Anderson's RBSes </div>
<br>1. Finish the alkaline lysis from Saturday 10.07, samples: 2.1, 3.1, 4.1, 5.1, 6.1
<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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<br>4. 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>All constructs are of the expected size.
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<br>
<div class="note">Cloning Connumity RBSes+GFP+terminator behind 23100 </div>
<div class="note">Cloning Connumity RBSes+GFP+terminator behind 23100 </div>
<br>2. Digest sample 12.1 J23100[synthetic promoter] with SpeI(Bcu)+ PstI,  
<br>2. Digest sample 12.1 J23100[synthetic promoter] with SpeI(Bcu)+ PstI,  
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<br>
<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
 
<div class="note">Cloning fluorescent proteins behind expression vector 3.1 and behind promoter J23100</div>
<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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<br>1. 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>2. Digest  fluorescent proteins with XbaI and PstI to clone into an expression vector. Fluorescent proteins:
 +
<br>- GFP E240
 +
<br>- CFP E0420
 +
<br>- YFP E0430
 +
<br>- mOrange E2050*
 +
<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
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<br>4. Gel extraction and ligation of fluorescent proteins with expression vector.
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<br>
 +
<br>
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<br>Electrophoresis (opis zdjęć wg kolejności naniesionych próbek):
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Gel 1: 2.1, 3.1, 4.1, 5.1, 6.1 [Anderson RBSes+GFPterminator]
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Gel 2: 7.2, 8.1, 9.1, 10.1 [Connumity RBSes+GFPterminator]
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Gel 3: 11.1, 12.1 [Community RBS+GFPterminator and J23100]
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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:

Revision as of 20:14, 13 July 2010

Example Tabs

Monday, 12 June 2010
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
4. 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.
Gel electrophoresis.[Fig X]
All constructs are of the expected size.
Cloning Connumity RBSes+GFP+terminator behind 23100

2. Digest sample 12.1 J23100[synthetic promoter] with SpeI(Bcu)+ PstI,
Gel electrophoresis [Fig X]
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
Gel electrophoresis {Fig X]
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
Cloning fluorescent proteins behind expression vector 3.1 and behind promoter J23100

1. Digest part part 3.1 – Anderson RBS from distribution (expression vector = promoter J23100+RBS) with SpeI and PstI
2. Digest fluorescent proteins with XbaI and PstI to clone into an expression vector. Fluorescent proteins:
- GFP E240
- CFP E0420
- YFP E0430
- mOrange E2050*
- mCherry J67102*
- mCherry NLS J130012
* can have an internal RBS. Check by cloning behind J23100.
3. Gel electrophoresis
4. Gel extraction and ligation of fluorescent proteins with expression vector.


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] Gel 4: GFP E240, CFP E0420, YFP E0430, mOrange E2050, mCherry J67102, mCherry NLS J130012
______________________________________________
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

blablabla temat2

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