Lab notes (8/9 - 8/15)

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:

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

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:
Over night cultures was grown from 4 colonies, until these OD’s was reached: Top 10 - no insert -> OD = 0,008 (100 x diluted)
Top 10 - with K274210 biobrick insert -> OD = 0,011 (100 x diluted)
MG1655 - no 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. all the over night cultures was grown for 20 hours at 37 °C. After 16 hours, ml of the over night cultures was transferred in to 110 LB media and grown for 4 hours to the exponential phase, until these OD’s was reached:
Top 10 - no insert -> OD = 0,049 (100 x diluted)
Top 10 - with K274210 biobrick insert -> OD = 0,044 (100 x diluted)
MG1655 - no 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:

The samples:

After this the standards and samples at the OD’s seen above, were measured on a UV-vis spectrometer. The stationary phase’s expression of beta-carotene by MG1655 E. coli and top 10 E. coli cells with standards solutions of beta-carotene, the graph 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:

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 was 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.