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Team:SDU-Denmark/labnotes14
From 2010.igem.org
Lab notes (10/11 - 10/17)
Contents |
Flagella Group
Transformation of FlhDCm composite part in pSB1C3 and pSB3K3 into Top 10 cells
Done By: Sheila and Louise
Date: 11/10 2010
Protocol: TR1.1
Notes:
Results:
Conclusion:
Retinal
Miniprep of K343005+DT in PSB1AK3
Date: 30/09
Done by: Marie
Methods: Miniprep
Protocols: MP1.1[1]
Notes:
marie skriver dette...
Restriction digest on K343002 and PSB1C3
Date: 12/10
Done by: Tommy
Methods: Restriction digest
Protocols: RD1.3[2]
Notes:
EcoRI and SpeI were used, the producted was purifided on a gel
nanodrop:
PSB1C3: 41,6
K343002: 64,5
Ligation of K343002 into PSB1C3
Date: 12/10
Done by: Marie & Christian
Methods: Ligation
Protocols: LG1.1[3]
Notes:
For each of the ligations three ligation mixtures were prepared. vector concentrations of 41,6ng/uL (PSB1C3) was used for mixture. Appropiate amount of insert was added to reach vector:insert ratios of 1:1, 1:2 and 1:3 respectively.
Ligation mixtures (B0015 in pSB3C5):
| L1 | L2 | L3 | |
| T4 ligase buffer | 2uL | 2uL | 2uL |
| T4 ligase | 1uL | 1uL | 1uL |
| pSB1C3 | 1uL | 1uL | 1uL |
| K343002 | 0.65uL | 1.30uL | 1.96uL |
| H20 | 34.35uL | 33,70uL | 33.04uL |
The samples was incubated at 17C ON at used for transformation
Transformations of K343002 in PSB1C3 into top10 E. Coli
Date: 12/10
Done by: Tommy
Methods: Ligation
Protocols: LG1.1[4]
Notes:
16 colonies and RFP control was selected from the colonie PCR. All of the colonies contain K343002 in PSB1C3
VF2 and VR primers was used
PCR Program:
| Start |
94 C |
2 min |
| Denaturing |
94 C |
1 min |
| Annealing |
55 C |
1 min |
| Elongation |
72 C |
2:30 min |
| Goto2 |
rep |
29x |
| End |
72 C |
3 min |
| Hold |
4 C |
Coloni PCR on transformations of K343002 in PSB1C3
Date: 30/09
Done by: Tommy
Methods: PCR
Protocols: CP1.3[5]
Notes:
Because the first coloni PCR of the transformants didn't give us the the right pieces 32 new colonies where chosen:
1-16: ninaB in PSB1C3
VF2 and VR used as primers in the TAQ PCR:
PCR Program:
| Start |
94 C |
2 min |
| Denaturing |
94 C |
1 min |
| Annealing |
55 C |
1 min |
| Elongation |
72 C |
2:30 min |
| Goto2 |
rep |
29x |
| End |
72 C |
2:30 min |
| Hold |
4 C |
