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Team:Warsaw/Calendar-Stage1/12 July 2010
From 2010.igem.org
Monday, 12 June 2010
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.
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
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
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
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