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Lab notes (8/9 - 8/15)

Contents 1 Lab notes (8/9 - 8/15) 1.1 Group: Photosensor 1.1.1 pfu PCR of B0015 and PCR purification 1.2 Group: Retinal 1.2.1 Characterization of Beta-carotene 1.2.1.1 Colony PCR on transformants using ninaB fwd and rw primers 1.2.1.2 Harvesting, sonication and UV-vis spektroscopy 1.2.2 PCR of NinaB (again) 1.2.2.1 Colony PCR on transformants using ninaB fwd and rw primers 1.2.3 Miniprep of POT2 with NinaB insert 1.2.3.1 ON of Top 10 E. coli cells with POT2 with NinaB 1.2.3.2 ON of Top 10 E. coli cells with POT2 with NinaB 1.2.3.3 Restriction digest on miniprep produckt (w. EcoRI)

Group: Photosensor

pfu PCR of B0015 and PCR purification

date: 17/8 Protocols: CP1.1 and GFX easy protocol
Notes:
2 uL PCR product of B0015 (no. 43 white) was used as template for each PCR reaction.3 PCR reactions were prepared.
Premix for 4 PCR reactions:

25uL	pfu bf. + MgSO4
7,5uL	dNTP's
7,5uL	VF2
7,5uL	VR
190uL	H20
2uL	pfu Polymerase enzyme

48uL premix is distrubuted into each PCR tube. PCR tubes are marked B0015.A-C
PCR program:

Start	95C	3min
Denaturating	95C	2min
Annealing	55C	30s
Elongation	72C	45s
Go to	2	29x
End	72C	2min
Hold	4C

5uL of PCR sample is loaded onto a 2% agarose gel. Generuler 100bp DNA ladder (blue) is used as marker.

Results:


Analysis: PCR product is OK and B0015 DNA is purified from the PCR product according to protocol. DNA is eluted in 20uL H2O. Purified samples are stored in the jumblebox (hotchpotch), marked as B0015-1 and B0015-2.

Group: Retinal

Characterization of Beta-carotene

Start date: 10/8
Methods: ON, sonication, UV-vis spectroscopy
Protocols: CC.1.1[1]

Colony PCR on transformants using ninaB fwd and rw primers

Date: 10/8
Done by: Christian & Tommy
Methods: ON
protocos:

Notes:
ON was made from 4 colonies:
Top 10 - on insert -> OD = 0,008 (100 x diluted)
Top 10 - with K274210 biobrick insert -> OD = 0,011 (100 x diluted)
MG1655 - on insert -> OD = 0,020 (100 x diluted)
Mg1655 - with K274210 biobrick insert -> OD = 0,017 (100 x diluted)
ON was made in 110 ml LB media. colonies with K274210 biobrick insert was grown in media containing ampicillin.
alle the ON cultures was grown for 20 hours at 37 °C.
After 16 hours, ml of the ON colonies was transferred in to 110 LB media and grown for 4 hours to the exponential phase:
Top 10 - on insert -> OD = 0,049 (100 x diluted)
Top 10 - with K274210 biobrick insert -> OD = 0,044 (100 x diluted)
MG1655 - on insert -> OD = 0,007 (100 x diluted)
Mg1655 - with K274210 biobrick insert -> OD = 0,009 (100 x diluted)
Colonies with K274210 biobrick insert was grown in media containing ampicillin.

Harvesting, sonication and UV-vis spektroscopy

Date: 11/8
Done by: Christian & Tommy
Methods: Cell harvesting, sonication and UV-vis messurements
protocos: none

Notes:
100 mL Cell cultures was centrifuged for 5 min at 14000 RPM. the supernantant was discarded and cells were resuspended in 5 mL acetone (99,9%), except the top 10 with the K274210 biobrick insert wicht was resuspended in 10 mL acetone (suorce of error.). The resuspended cells were sonicated for 5 min. the samples were spun down and the supernantant were transferrede to new tubes, cell bebri was discarded.
A standart curve was made from pure Beta-carotene,
The samples and the standarts were first measured at a fixed wavelengt of 456 nm. The standarts:

Concentration	Absorbance
1 mM	4,000
100 µM	2,260
50 µM	4,000
25 µM	0,155
10 µM	0,893
5 µM	0,440
1 µM	0,075
100 nM	0,015
10 nM	0,038
1 nM	0,005
100 pM	0,024

The samples:

	Top 10 cells (Absorbance)	MG1655 E. coli mutant (Absorbance)
Stationary phase control	0,034	0,024
Stationary phase with K274210 biobrick insert	0,319	1,549
Expotential phase control	0,020	0,024
Expotential phase with K274210 biobrick insert	0,034	0,033

After this the standarts and samples were measured on a UV-vis spetrometer. The stationary phases expersion of beta-carotene by wild strain MG1655 E. coli and top 10 E. coli cells with standarts solutions of beta-carotene, the graf is shown below

PCR of NinaB (again)

Start date: 13/8
Methods: Restriction digest, PCR, gel
Protocols: RD1.1 [2], CC.1.1[3]

Colony PCR on transformants using ninaB fwd and rw primers

Date: 13/8
Done by: Marie & Tommy
Methods: Restriction digest, PCR, gel
protocos: RD1.1, CC.1.1

Notes:
Restirction digest was performed with EcoRI according to protocol. (gel was run on protocol).
PCR was run on the product (No gel purification).
The following PCR program was used:

PCR	Temp. (C)	Time
Start	95	2 min
Denaturation	95	45 sec
Annealing	49	30 sec
Elongation	72	4 min
Denaturing	95	45 sec
Annealing	69	30 sec
Elongation	72	4 min
End	72	5 min
Hold	4	indef.

gel was run on the PCR product.

Miniprep of POT2 with NinaB insert

Start date: 13/8
Methods: Miniprep, Restriction digest, gel
Protocols: MP1.2 [4], RD1.1 [5].

ON of Top 10 E. coli cells with POT2 with NinaB

Date: 16/8
Done by: Marie & Tommy
Methods: protocos:

Notes:
100 mL ON culture was made, cells were grown at 37 C in LB media with Chloramphenicol.

ON of Top 10 E. coli cells with POT2 with NinaB

Date: 17/8
Done by: Marie & Tommy
Methods: Miniprep, Restriction digest, gel
Protocols: MP1.2 [6], RD1.1 [7].

Notes:
50 mL of elution buffer was used. DNA concentrations after pooling were measured on NanoDrop to 206,2 ng/microL. gel was run on produckt, showing recent miniprep compared to old miniprep produckt, showing uneven lengths. subsequent wa performed.

Restriction digest on miniprep produckt (w. EcoRI)

Date: 17/8
Done by: Marie & Tommy
Methods: Restriction digest, gel
Protocols: MP1.2 [8], gel.

Notes:
Due to the lack of old sample, restriction digest was performed using only 3,5 microL of miniprep produckt. 1,5 microL of H2O was added insted. EcoRI was used

The digest mix was incubated for 10 min at 37 C. A gel was run showing uncut new, cut new, uncut old and cut old miniprep product.