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Team:Warsaw/Calendar-Stage1/12 July 2010
From 2010.igem.org
Monday, 12 June 2010; summarised by Milena
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.
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]
______________________________________________
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
RBSes+GFPterminator at expected size, vectors at expected size;
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
______________________________________________
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 [Community RBSes+GFPterminator]
RBSes+GFPterminator at expected size, vectors at expected size;
