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Lab notes (10/11 - 10/17)

Contents 1 Lab notes (10/11 - 10/17) 1.1 Flagella Group 1.1.1 Transformation of FlhDCm composite part in pSB1C3 and pSB3K3 into Top 10 cells 1.2 Retinal 1.2.1 Miniprep of K343005+DT in PSB1AK3 1.2.2 Restriction digest on K343002 and PSB1C3 1.2.3 Ligation of K343002 into PSB1C3 1.2.4 Transformations of K343002 in PSB1C3 into top10 E. Coli 1.2.5 Coloni PCR on transformations of K343002 in PSB1C3

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

Colonies 1, 2, 3 and 4 was selected for minipreps and freezing cultures.