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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 (FlhDCmut) 1.1.2 Amplification of FlhDCmut 1.1.3 Gel extraction of FlhDCmut 1.1.4 Restriction digest of FlhDCmut and pSB1C3 1.1.5 Gel extraction of FlhDCmut and pSB1C3 1.1.6 Amplification of FlhDCmut 1.1.7 Gel extraction of FlhDCmut 1.1.8 Gel extraction of flhD/C (native coding sequence) 1.1.9 Digestion of flhD/C (native coding sequence) and pSB1A2 with EcoRI SpeI 1.1.10 Gel extraction of digested flhD/C (native coding sequence) and pSB1C3 1.1.11 Ligation of flhD/C (native coding sequence) and pSB1C3 1.1.12 Coloni PCR of flhD/C in pSB1C3 1.1.13 Coloni PCR of flhD/C in pSB1C3 1.1.14 Digestion of flhD/C PCR product with EcoRI and SpeI 1.1.15 Purification of pSB1A2 containing J13002 1.1.16 Amplification of pSB1A2 w. J13002, pSB1A2 w. B0015 and pSB1A2 w. B0034 1.2 Group: Photosensor 1.2.1 Miniprep of pSB3T5 w. J04450 and pSB3C5 w. J04450 from transformation 20/7 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 1.3.3 Coloni PCR of R0011 in pSB1A2 and K081005 in pSB1A2 1.3.4 Miniprep of pSB3T5 w. J04450 and pSB3C5 w. J04450 from transformation 20/7 1.3.5 Transformation of TOP 10 e. coli with ninaB gene in pOT2:chlor vector 1.3.5.1 Transformation of cells with gene in plasmid 1.3.6 Coloni PCR of flhD/C in pSB1C3

Group: Flagella

Annealing of the two mutated strands of FlhDC (FlhDCmut)

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

Pfu buffer	5,0uL
dNTP	1,5uL
FlhDC mutated long strand	1,5uL
FlhDC mutated short strand	1,5uL
H2O	41,0uL
Pfu	1,0uL

The samples were run in the following PCR program:

Program	1st sample	2nd sample	Time
1: Start	94˚C	94˚C	3min
2: Denaturing	94˚C	94˚C	2min
3: Annealing	56,1˚C	64,5˚C	30sec
4: Elongation	72˚C	72˚C	2min
5:	GOTO	Repeat	X29
6: End	72˚C	72˚C	2min
7: Hold	4˚C	4˚C

Results: A gel, containing the PCR product was run, using [http://www.fermentas.de/product_info.php?info=p1110 GeneRuler(TM) DNA Ladder Mix, ready-to-use (#SM0333)] loaded in lane one (from the left:


We can se very faint bands in lane 2 and 3, at around 1000bp. The band in lane 3 is a little more visible than lane 2. Because we haven't used primers ind this PCR, it is possible that the two mutated strands has a lower possibility of annealing to each other. It s however possible to amplify the few complete FlhDCmut strands in a new PCR, which is what we will do next.

--Sheila 09:10, 22 July 2010 (UTC)

Amplification of FlhDCmut

Experiment done by: Sheila

Date: July 19th

Protocol: CP1.1

Method: The experiment was done in two rounds. First, the annealing product, made by Pernille on july 19th was amplified. The we amplified Pernilles amplification simultaneously as we amplified Sheilas previous annealing.
Primers used: FlhDC fw and FlhDC rev
Polymerase used: Pfu

Notes:

Results:

Gel extraction of FlhDCmut

Experiment done by: Sheila

Date: July 20th

Protocol:

Method:

Notes:

Results:

Restriction digest of FlhDCmut and pSB1C3

Experiment done by: Sheila

Date: July 20th

Protocol:

Method:

Notes:

Results:

Gel extraction of FlhDCmut and pSB1C3

Experiment done by: Sheila

Date: July 20th

Protocol:

Method:

Notes:

Results:

Amplification of FlhDCmut

Experiment done by: Sheila

Date: July 20th-21st

Protocol: CP1.1

Method:

Notes:

Results:

Gel extraction of FlhDCmut

Experiment done by: Sheila

Date: July 21st

Protocol:

Method:

Notes:

Results:

Gel extraction of flhD/C (native coding sequence)

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
flhDC 1	19.82	2.43	0.08
flhDC 2	25.24	2.22	0.12

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

Digestion of flhD/C (native coding sequence) 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 (native coding sequence) 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 (native coding sequence) 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
surplus ligation mixture was stored at -20 degrees as L1, L2 and L3.
--Tipi 18:15, 19 July 2010 (UTC)

Coloni PCR of flhD/C in pSB1C3

Start date: 21/07 End date: 21/07
Methods: Coloni PCR and gel electrophoresis

Protocol:CP1.2

Experiment done by: Maria and LC

Notes:
Coloni PCR was made on flhD/C in pSB1C3 from Transformation.
10 colonies from plates plated with cells transformed with L2 and L3 ligation mixtures (see Ligation).
Coloni 1-5 is taken from L2 plates coloni 6-10 is taken from L3 plates
PCR was made with only half the amount of premix.
Premix for 12 PCR reactions:

10xtaq buffer	30uL
MgCl2	12uL
VF2	12uL
VR	12uL
dNTP	6uL
H2O	42uL
Total vol.	114uL

0.5uL Taq polymerase from ampliqon was used.
Taq PCR program:

1:Start	94C	2 min
2: Denaturing	94C	1 min
3: Annealing	55C	1 min
4: Elongation	72C	1 min 30 s
5:	GO TO	rep. 29x
6: End	72C	3 min
7: Hold	4C

PCR product was loaded onto a 2% agarose gel. Gene ruler DNA ladder mix (red) was used as marker.

VF2 - coding sequence (in pSB1C3) : 140bp
Coding sequence - VR (in pSB1C3)  : 176bp
flhD/C  : 932bp
VF2 - VR for pSB1C3 w. flhD/C  :1248bp

VF2 - VR for pSB1C3 without insertion : 316bp

Results:
Gel electrophoresis:

Analysis:
All lanes except 4, 9 and 10 showed a band at app. 1200bp. which might indicate plasmids where flhD/C has been inserted. Lane 4 and 9 has a band at app. 1500bp which could indicate undigested pSB1C3 (VF2 - VR for pSB1C3 w. J04450 is 1385bp).
Streak plates of coloni 1, 2, 3, 5, 6, 7, 8 and 10 showed red colonies, indicating that these contained undigested pSB1C3.
Streak plates of coloni 4 and 9 had only white colonies, suggesting an insertion has occured.
To verify if flhD/C has been inserted coloni PCR using VF2 and flhD/C rev. as primers was carried out for colonies from streak plates of coloni 4 and 9 and of all white colonies from the transformation plates. (see coloni PCR)
--Tipi 11:46, 24 July 2010 (UTC)

Coloni PCR of flhD/C in pSB1C3

Start date: 22/07 End date: 22/07
Methods: Coloni PCR and gel electrophoresis

Protocol:CP1.3

Experiment done by: Maria

Notes:
Coloni PCR was made on flhD/C in pSB1C3 from Transformation.
All remaining white colonies from plates plated with cells transformed with L2 and L3 ligation mixtures (see Ligation) was selected.
coloni 1-6 is taken from L2.3 plates
coloni 7-15 is taken from L3.2 plates
coloni 16-27 is taken from L3.3 plates
coloni 28 is taken from streak plate of PCR no. 4 coloni 29 is taken from streak plate of PCR no. 9 (see Coloni PCR)
Premix for 31 PCR reactions:

10xtaq buffer	77.5uL
MgCl2	31uL
VF2	31uL
flhD/C rev.	31uL
dNTP	15.5uL
H2O	108.5uL
Total vol.	294.5uL

0.5uL Taq polymerase from fermentas was used.
Taq PCR program:

1:Start	94C	2 min
2: Denaturing	94C	1 min
3: Annealing	55C	1 min
4: Elongation	72C	1 min 30 s
5:	GO TO	rep. 29x
6: End	72C	3 min
7: Hold	4C

PCR product was loaded onto a 2% agarose gel. Gene ruler DNA ladder mix (red) was used as marker.

Results:
Gel electrophoresis:

Analysis:
None of the samples contained amplified DNA.
This indicates either that the flhD/C gene has not been inserted to the pSB1C3 backbone or an insertion has been made but the flhD/C primer has not been able to anneal to the template.
To verify that a ligation has occurred, PCR product 4 and 9 from Coloni PCR was digested with EcoRI and SpeI.
--Tipi 13:53, 24 July 2010 (UTC)

Digestion of flhD/C PCR product with EcoRI and SpeI

Start date: 24/07 End date: 24/07
Methods: Restriction digest and gel electrophoresis

Protocol:RD1.1

Experiment done by: Maria

Notes:
To verify if a ligation has occurred Taq PCR product of coloni 4 and 9 (see Coloni PCR) was digested with the same restiction enzymes used for the digestion of flhD/C DNA and pSB1C3 prior to ligation.
If an ligation has occurred, then the restriction sites of EcoRI and SpeI should be intact, and two fragments of 122bp and 170bp should be observed on the gel, indicating VF2-E site and S site-VR respectively.
RFP (PCR poduct of coloni no.8) was used as controle.
2.5uL of undigested PCR product of coloni 4, 8 and 9 were loaded onto the gel as well.
Samples were loaded onto a 2% agarose gel. Hyper Ladder II was used as marker.
Loading scheme:

Lane	1	2	3	4	5	6
	digested no. 4	undigested no. 4	digested no. 9	undigested no. 9	digested no. 8	undigested no. 8

Results:
Gel electrophoresis:


Analysis:
The 2 bands at app. 100bp and 200bp respectively indicates that the E and S restiction sites have been preserved, suggesting that a ligation has occured.
An ON culture of coloni 4 and 9 is made to use for mini-prep.
--Tipi 14:37, 24 July 2010 (UTC)

Purification of pSB1A2 containing J13002

Experiment done by: Pernille

Date: the 19 of July

Protocol: MP1.1 and CP1.1

Method: Miniprep, Nano drop, gel electrophoresis and PCR

Notes: The pSB1A2 plasmid are a high-copy-plasmid and therefore 5mL ON culture are used for purification of the plasmid. for the PCR the following PRC program are used: 1: 94°C 3min 2: 94°C 2min 3: 55°C 30 sec 4: 72°C 1min 5: x 29 rep. 6: 72°C 5min 7: hold 4°C

Results:After the purification the concentration of nucleic acid concentration was measured on the Nano Drop. There was made four purification tubes. Only tube 1 and 3 was used for further experiments because the other contained a very low concentration of nucleic acid. The concentration in tube 1 and 3 was 25,72ng/uL and 25,76ng/uL. The purified DNA was loaded in a 1,5% agarose gel together with a 10.000bp long ladder (red).

PCR was run on the DNA using the VR and VF2 primers wich ensures the only the brick part are amplificated. The pfu polymerase was used but unfortunately i did not get any results since i did not add any MgSO4 to the premix.

--Pernm07 08:48, 22 July 2010 (UTC)

Amplification of pSB1A2 w. J13002, pSB1A2 w. B0015 and pSB1A2 w. B0034

Experiment done by: Pernille

Date: The 20th of July

Protocol: CP1.1and DNA extraction

Method: PCR and DNA extraction

Notes: The purified DNA was amiplificated with PRC using the following program: 1: 94°C 3min 2: 94°C 2min 3: 55°C 30 sec 4: 72°C 1min 5: x 29 rep. 6: 72°C 5min 7: hold 4°C

the experiment for each biobrick was do in duplicats.

Results:After amplification the nucleic acid concentration was measured on the Nano Drop. The concentrations was measued to:

tube 1: pSB1A2 w. J13002 was 825.2ng/uL tube 2: pSB1A2 w. B0015 was 547.76ng/mL tube 3: pSB1A2 w. B0034 was 777.84ng/uL

The PCR product was loaded on a 2% agarose gel together with a 1000 bp long marker. xxxxxxxxxx

During the DNA extraction 50uL DNA anf 7uL loading buffer was loaded in a lane. The samples was loaded from left to right in continuous numbers. xxxxxxx The concentration of the extracted DNA from tube 1,2 and was found to 13.60ng/uL, 16,58ng/uL and 12,6313.60ng/uL respectively. while we have to use the dobbelt terminator and the constitutive active promotor in a lot of experiment the next step is to do another round of PRC and DNA extraction on thise biobricks.

--Pernm07 10:06, 22 July 2010 (UTC)

Group: Photosensor

Miniprep of pSB3T5 w. J04450 and pSB3C5 w. J04450 from transformation 20/7

Start date: 22/07 End date: 22/07
Methods: Miniprep, gel electrophoresis and nano drop

Protocol:MP1.2

Experiment done by: LC

Notes: /

Results:
Gel electrophoresis:

Nanodrop:
The concentration was around 23 ng/ul for all samples. Not overwhelming, but usable.

Analysis:
Sample 1-4 of BBa_K081005 were pooled and frozen and sample 1-4 of BBa_R0011 were too.

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:
Both pSB3T5 and pSB3C5 was succesfully transformed, and ON cultures with appropiate antibiotics were made for mini-prep.
For pSB1A2 w. R0011 only 6 colonies was observed on the LA+amp plate.
For pSB1A2 w. K081005 only 4 colonies was observed on the LA+amp plate.
All 10 colonies were used for Coloni PCR.
--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.

Results:

Analysis:
Test plasmid was succelsfully transformed and we can carry on transforming the plasmid containing the NinaB gene.
Colonies was seen on plates transformed with L2 and L3. 10 colonies were selected and used for coloni PCR.
--Tipi 08:17, 21 July 2010 (UTC)

Coloni PCR of R0011 in pSB1A2 and K081005 in pSB1A2

Start date: 20/07 End date: 20/07
Methods: Coloni PCR and gel electrophoresis

Protocol:CP1.2

Experiment done by: Maria and LC

Notes:
6 colonies transformed with pSB1A2 w. R0011 and 4 colonies transformed with pSB1A2 w. K081005 (see Transformation) was used for coloni PCR.
Colonies with K081005 was denoted A1, A2 A3 and A4 respectively.
Colonies with R0011 was denotes B1, B2, B3, B4, B5 and B6 respectively
PCR was made with only half the amount of premix.
Premix for 12 PCR reactions:

10xtaq buffer	30uL
MgCl2	12uL
VF2	12uL
VR	12uL
dNTP	6uL
H2O	42uL
Total vol.	114uL

0.5uL Taq polymerase from ampliqon was used.
Taq PCR program:

1:Start	94C	2 min
2: Denaturing	94C	1 min
3: Annealing	55C	1 min
4: Elongation	72C	30 s
5:	GO TO	rep. 29x
6: End	72C	3 min
7: Hold	4C

PCR product was loaded onto a 2% agarose gel. Gene ruler 100bp DNA ladder was used as marker.

Results:
Gel electrophoresis:


Analysis:
A band at app. 300bp was observed in all lanes, indicating that all 10 colonies contained the correct plasmids.
ON cultures were made for mini-prep.
--Tipi 06:35, 21 July 2010 (UTC)

Miniprep of pSB3T5 w. J04450 and pSB3C5 w. J04450 from transformation 20/7

Start date: 21/07 End date: 21/07
Methods: Miniprep, gel electrophoresis and nano drop

Protocol:MP1.2

Experiment done by: Maria and LC

Notes:
For pSB3T5 no.3 one half of the material was lost during the neutralization step.
pSB3T5 no.1 was eluted in 200uL elution buffer.
Samples were loaded onto a 1.5 agarose gel. Generuler DNA ladder-mix (red) was used as marker

Results:
Gel electrophoresis:

Nanodrop:

Sample ID	ng/uL	260/280	260/230
pSB3C5 1	208.2	1.93	2.31
pSB3C5 2	225.09	1.93	2.3
pSB3C5 3	215.2	1.93	2.3
pSB3C5 4	260.68	1.93	2.33
pSB3T5 1	14.16	2.06	2.0
pSB3T5 2	52.50	1.96	2.15
pSB3T5 3	26.45	1.99	2.3
pSB3T5 4	53.43	2.0	2.19

Analysis:
Both pSB3T5 and pSB3C5 was succesfully purified.
pSB3C5 1-4 was pooled and stored as no.28 (white) at -20 degrees.
pSB3T5 1+3 was pooled and stored as no.30 (white) at -20 degrees.
pSB3T5 2+4 was pooled and stored as no.29 (white) at -20 degrees.

Nanodrop of pooled samples:

Sample ID	ng/uL	260/280	260/230
pSB3C5 1-4	256.71	1.92	2.3
pSB3T5 1+3	54.01	1.95	2.19
pSB3T5 2+4	16.0	1.88	2.11

--Tipi 09:41, 21 July 2010 (UTC)

Transformation of TOP 10 e. coli with ninaB gene in pOT2:chlor vector

Date: 21/7
Done by: Christian

Transformation of cells with gene in plasmid

Date: 21/7
Methods: Transformation
Protocols: TR1.1 and paperblotter protocol from plasmid cDNA package.

Notes: Filterpaper was washed with 50ul TE buffer for 2 seconds. Paper punch was then transfered to a sterile eppendorff tube, and transformation protocol TR1.1 was followed from here. The usual positive control was used.

Plates were dried for ca. 12 minutes, showed no sign of cracking.

The gene is inserted into a pOT2 plasmid. A plasmid map can be found [http://www.fruitfly.org/about/methods/pOT2vector.html here]:

Two of the chlor plates were damaged (one very slightly) and one ampicilin plate was torn up, again. (good thing two were dried). Plates were incubated ON at 37°.

Coloni PCR of flhD/C in pSB1C3

Start date: 21/07 End date: 21/07
Methods: Coloni PCR and gel electrophoresis

Protocol:CP1.2

Experiment done by: Maria and LC

Notes:
Coloni PCR was made on flhD/C in pSB1C3 from Transformation.
10 colonies from plates plated with cells transformed with L2 and L3 ligation mixtures (see Ligation).
Coloni 1-5 is taken from L2 plates coloni 6-10 is taken from L3 plates
PCR was made with only half the amount of premix.
Premix for 12 PCR reactions:

10xtaq buffer	30uL
MgCl2	12uL
VF2	12uL
VR	12uL
dNTP	6uL
H2O	42uL
Total vol.	114uL

0.5uL Taq polymerase from ampliqon was used.
Taq PCR program:

1:Start	94C	2 min
2: Denaturing	94C	1 min
3: Annealing	55C	1 min
4: Elongation	72C	1 min 30 s
5:	GO TO	rep. 29x
6: End	72C	3 min
7: Hold	4C

PCR product was loaded onto a 2% agarose gel. Gene ruler DNA ladder mix (red) was used as marker.

VF2 - coding sequence (in pSB1C3) : 140bp
Coding sequence - VR (in pSB1C3)  : 176bp
flhD/C  : 932bp
VF2 - VR for pSB1C3 w. flhD/C  :1248bp

VF2 - VR for pSB1C3 without insertion : 316bp

Results:
Gel electrophoresis:

Analysis:
All lanes except 4, 9 and 10 showed a band at app. 1200bp. which might indicate plasmids where flhD/C has been inserted. Lane 4 and 9 has a band at app. 1500bp which could indicate undigested pSB1C3 (VF2 - VR for pSB1C3 w. J04450 is 1385bp).

Include column content here.