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Lab notes (7/19 - 7/25)

Contents 1 Lab notes (7/19 - 7/25) 1.1 Group: Flagella 1.1.1 Annealing of the two mutated strands of FlhDC 1.1.2 Gel extraction of flhD/C 1.1.3 Digestion of flhD/C and pSB1A2 with EcoRI SpeI 1.1.4 Gel extraction of digested flhD/C and pSB1C3 1.1.5 Ligation of flhD/C and pSB1C3 1.2 Group: Photosensor 1.3 Group: Retinal 1.3.1 Transformation of K081005 in pSB1A2 (constitutive promoter and RBS combined),R0011 in pSB1A2, pSB3C5 w. J04450 and pSB3T5 w. J04450 in Top 10 E.Coli 1.3.2 Transformation of flhD/C in pSB1C3 and test plasmid in Top 10 E.Coli

Group: Flagella

Annealing of the two mutated strands of FlhDC

Experiment done by: Sheila

Date: July 19th

Protocol: CP1.1

Method: PCR of the two mutated strands of the FlhDC operon

Notes: To samples were run at two different temperatures: 56,1˚C and 64,5˚C respectively.
Polymerase used: Pfu
Primers used: None, as the two strands are supposed to anneal to each other

Results:

Gel extraction of flhD/C

Start date: 19/07 End date: 19/07
Methods: Gel extraction, nanodrop
Protocol:DE1.1
Experiment done by:Maria

Notes:
70uL of flhD/C PCR product from Amplification of flhD/C was loaded onto a 1.5 agarose gel and extracted according to protocol.
DNA was eluted in 20uL elution buffer.

Results:
Nanodrop:

Sample ID	ng/uL	260/280	260/230
flhD/C 1	19.82	2.43	0.08
flhD/C 2	25.24	2.22	0.12

Analysis:
Nanodrop measurements idicated a possible contamination. However the DNA was pooled and used for Digestion
--Tipi 17:22, 19 July 2010 (UTC)

Digestion of flhD/C and pSB1A2 with EcoRI SpeI

Start date: 19/07 End date: 19/07
Methods: Digestion, Gel electrophoresis
Protocol:RD1.1
Experiment done by:Maria

Notes:
purified pSB1C3 (tube 18 blue) and flhD/C from Gel extraction was digested with EcoRI and SpeI.
Restriction mixture:

	flhD/C	pSB1C3
H2O	30uL	24uL
EcoRI	4uL	2uL
SpeI	4uL	2uL
FD green buffer	8uL	4uL
DNA	38uL	10uL
total vol.	84	42

samples were loaded onto a 1.5 agarose gel. Gene ruler red were used as marker.

Results:
Gel electrophoresis:


Analysis:
In lane 2 containing pSB1C3 2 bands are detected indicating a succesful digestion of the plasmid (the band at 1000 bp corresponds to J04450). A succesful digestion of the flhD/C cannot be concluded from the gel. However both bands was excised and extracted from gel (Gel extraction)
--Tipi 17:42, 19 July 2010 (UTC)

Gel extraction of digested flhD/C and pSB1C3

Start date: 19/07 End date: 19/07
Methods: Gel extraction, nanodrop
Protocol:DE1.2
Experiment done by:Maria

Notes:
Digested flhD/C and pSB1C3 from Digestion were extracted from gel according to protocol.
DNA was eluted in 20uL H20.

Results:
Nanodrop:

Sample ID	ng/uL	260/280	260/230
flhD/C	18.18	4.59	0.18
pSB1C3	14.30	1.89	0.04

Analysis:
nanodrop measurements indicated contamination. However both samples were used for Ligation.
--Tipi 17:57, 19 July 2010 (UTC)

Ligation of flhD/C and pSB1C3

Start date: 19/07 End date: 20/07
Methods: Ligation
Protocol:LG1.2
Experiment done by:Maria

Notes:
3 ligation reactions was prepared.

	LG1	LG2	LG3
10x T4 ligase buffer	2uL	2uL	2uL
flhD/C	5uL	5uL	5uL
pSB1C3	1uL	2.8uL	5uL
H20	11uL	9.2uL	7uL
T4 ligase	1uL	1uL	1uL

Ligation mixtures were not run on gel but were directly used for transformation
--Tipi 18:15, 19 July 2010 (UTC)

Group: Photosensor

Group: Retinal

Transformation of K081005 in pSB1A2 (constitutive promoter and RBS combined),R0011 in pSB1A2, pSB3C5 w. J04450 and pSB3T5 w. J04450 in Top 10 E.Coli

Start date: 19/07 End date: 20/07
Methods: ON culture, making competent cells, transformation

Protocol:CC1.1 TR1.1

Experiment done by: Maria

Notes:ON colony was made of 110 ml lb medium inoculated with a top10 coloni.

Time	Optical  density
8:12	2.9
8:17	0.02
9:17	0.035
10:17	0.204
10:40	0.380
10:50	0.49

pSB1A2 w. R0011 and pSB1A2 w. K081005 was plated with 150uL on plates containing LA, LA + Amp, LA + Tetracycline, LA + Chloramphenicol and LA + Kanamycine.Upconcentration of these samples was not made. pSB3T5 w. J04450 and pSB3C5 w. J04450 was plated according to protocol

Results:

Analysis:
--Tipi 16:33, 19 July 2010 (UTC)

Transformation of flhD/C in pSB1C3 and test plasmid in Top 10 E.Coli

Start date: 20/07 End date: 19/07
Methods: ON culture, making competent cells, transformation

Protocol:CC1.1 TR1.1

Experiment done by: Maria

Notes:
Ligated flhD/C from Ligation and test plasmid from Whatman was transformed.
ON colony was made of 25 ml lb medium inoculated with a top10 coloni.

Time	Optical  density
8:17	3.5
8:20	0.02
9:15	0.035
10:30	0.222
10:52	0.350
11:05	0.52

Compotent cells were washed using 10mL 50mM CaCl2.
3 parallel transformations were carried out for L1, L2 and L3 respectively (see Ligation). L1.1, L1.2, L2.1, L2.2 and L3.1 were transformed using compotent cells from 19/7 (Compotent cells).
Prior to transformation test plasmid was washed with 10xTE pH 8.0.
--Tipi 15:19, 20 July 2010 (UTC)

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