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Team:SDU-Denmark/labnotes5
From 2010.igem.org
Lab notes (8/9 - 8/15)
Contents |
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: CP.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 (ON) cultures were grown from 4 colonies, until the following OD’s were obtained:
Top 10 - no insert OD = 0,008 (100 x diluted)
Top 10 - with K274210 insert OD = 0,011 (100 x diluted)
MG1655 - no insert OD = 0,020 (100 x diluted)
Mg1655 - with K274210 insert OD = 0,017 (100 x diluted)
ON cultures were grown in 110 ml LB media. Colonies with K274210 insert were grown in LB media containing ampicillin. All ON cultures were grown for 20 hours at 37 °C. After 16 hours, 10 ml of the ON cultures were transferred into 110 ml LB media and grown for 4 hours to reach the exponential phase, where the following OD’s were obtained:
Top 10 - no insert OD = 0,049 (100 x diluted)
Top 10 - with K274210 insert OD = 0,044 (100 x diluted)
MG1655 - no insert OD = 0,007 (100 x diluted)
Mg1655 - with K274210 insert OD = 0,009 (100 x diluted)
Cultures with K274210 insert were grown in media containing ampicillin.
100 mL cell culture were centrifuged for 5 min at 14000 RPM. The supernatant was discarded and cells were resuspended in 5 mL acetone (99,9%), except the Top 10 E. coli with the K274210 insert, which was resuspended in 10 mL acetone (source of error). The resuspended cells were sonicated for 5 min. Samples were spun down, the supernatant was transferred to new tubes, and cell debris was discarded. A standard curve was made from pure beta-carotene.
The samples at the OD’s seen above as well as solutions of pure beta-carotene with known concentrations were measured at a fixed wavelength of 456 nm. Known concentrations and their absorbances:
| 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 and their absorbances:
| 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 |
UV-VIS absorbance spectra of the known solutions were obtained, as well as spectra of the samples at the ODs shown above. The spectra of Top 10 and MG1655 in the stationary phase as well as selected spectra of known solutions are shown below:
PCR of NinaB (again)
Start date: 13/8
Methods: Restriction digest, PCR, gel
Protocols: RD1.1 [1], CC.1.1[2]
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
