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__august
08-08-2008
The T-cells B12.7.5 from Mahima
10 ml of the cells were taken from the culture (30 ml) and centrifuged at 1200 rpm for 5 minutes. Then the old medium was replaced by 10 ml of fresh medium, the cells were resupended and transferred to a new dish with 20 ml fresh medium.
The cells have to be split approximately every 3 days (the medium should start to become yellow).
We use RPMI medium containing 10% FCS, HEPES 10mM, ß-Mercaptoethanol 50µM, L-Glutamine 2mM and Pen/Strep 1%.
08-11-2008
Solving the parts of the IGEM 2008 parts collection and transformation
I warmed 5 µl TE to 50° in PCR tubes. Then I punched out the desired parts (CMV promotor BBa_I712004, transfectionvector BBa_J52017) and put it into the warmed TE puffer, spin the tubes and after 20 minutes I started the transformation with the following protocol
- 2µl of the eluted DNA to the thawed competent cells (RV308)
- 20 minutes on ice
- heat shock 42° for 1 minute
- 5 minutes on ice
- 900µl DyT and incubation at 37° for 70 minutes
- plating out of 50µl and 100µl on LB/Amp plates (1µl Amp stock solution for 1 ml LB)
08-12-2008
Solving the parts of the IGEM 2008 parts collection and transformation
There were no colonies on the plates so I changed the protocol and tried it again.
I solved the DNA in 10µl warmed TE and incubated at 50° while elution. And the transformation:
- 5µl of the eluted DNA to XL-1 cells
- 30 minutes on ice
- heat shock 42° 1 minute
- 5 minutes on ice
- 900µl DyT and transfering the cells in bigger tubes for a better ventilation for 2 hours at 37°
- plating out of 50µl on LB/Amp plates
- centrifuging for 3 minutes at 1000 rpm
- put away the supernatant and plating out the remaining 100µl
Freezing aliquots of the B.12.7.5 cells from Mahima
- mixing FCS with 15% DMSO
- centrifuging 30ml of the cell suspension 5 minutes at 1200rpm
- taking away the old medium and resuspending the cells in 2.5 ml FCS/DMSO
- making 0.5ml aliquots in special freezing tubes
- freezing at -80°
08-13-2008
There again were no colonies on the plates.
08-14-2008
The freezed aliquots of the B.12.7.5 cells
I put the cells in nitrogen. They are located in the tank 1, stock 5, box 6 (the first row with the red caps)
Using the part collection of 2007
- 15µl ddH20 to the wells of the sc-fluorescein(BBa_J07014) and the transfectionvector(BBa_J52017)and puting in an 1.5ml Eppi
- 1µl to XL-1 cells
- 20 minutes on ice
- 1 minute heat shock 42°
- 5 minutes on ice
- 900µl DyT and transfering to bigger tubes
- 70 minutes at 37°
- plating out 50µl on LB/Amp plates and centrifuging the reamining cells at 1000rpm for 3 minutes
- discarding the supernatant and plating out the remaining 100µl
08-15-2008
There were colonies on the plates from both parts.
08-17-2008
Picking colonies
I picked three colonies from each plate (sc-fluorescein and transfectionvector), put them into 5ml LB/Amp and incubated at 37° ~16h
Transformation of the parts CMV promotor(BBa_J52034) and CMV+Rluc(BBa_J52038) from 2007
I did the transformation using the protocol from "Using the part collection of 2007"(08-14-2008)
08-18-2008
Miniprep of the picked colonies (sc-fluorescein and transfectionvector)
I did the Miniprep with the QIAprep Spin MIniprep Kit
Analytic digestion
For the sc-fluorescein I did a double digestion with SpeI and EcoRI:
- 5µl plasmid, 0,5µl SpeI, 0,5µl EcoRI, 0,5µl BSA, 1µl Buffer P2, 2,5µl H2O
- 2,5h at 37°
- 1% agarose gel
For the transfectionvector I did a digestion with EcoRI:
- 5µl plasmid, 1µl EcoRI, 1µl Buffer P2, 3µl H2O
- 2,5h at 37°
- 1% agarose gel
Picking colonies
I picked three colonies from each plate (CMV promotor and CMV+Rluc), put them into 5ml LB/Amp and incubated for ~16h at 37°
08-19-2008
Analytic digestion
For the CMV promotor and the CMV+Rluc construct I did a double digestion with SpeI and EcoRI:
- 5µl plasmid, 0,5µl SpeI, 0,5µl EcoRI, 0,5µl BSA, 1µl Buffer P2, 2,5µl H2O
- 2,5h at 37°
- 1% agarose gel
08-22-2008
Thawing 293T cells
For expressing our receptor constructs we decided to use 293T cells. We wanted to test the efficiency of transfection of the 293T cells, so I thawed the cells and put them in culture. I used DMEM medium with 1% Glutamine, 10%FCS and 1% Pen/Strep. I thawed the cells at 37°C and put them in a cell culture flask with 20 ml medium
08-25-2008
Splitting the cells
I split the cells and transfered them to a 6 well dish. You should transfer 6*10^4 cells/cm². I counted the cells in the neubauer chamber and counted 15*10000 cells/ml so I transfered 500 µl of the cell suspension in one well.
08-26-2008
Transfection
One hour before transfection I washed the cells once with PBS and filled up with fresh medium. Normann did the transfection with a plasmid carrying the ß-Galactosidase gene.
08-28-2008
Harvesting cells
For the ONPG Test we decided to use cells after several timepoints. So I harvested cells after 48h.
I washed the cells twice with PBS. Then I took 500µl PBS, lost the cells with a cell scraper and transfered it to an eppi. After centrifugation (2min 13000rpm) I replaced the PBS with 500µl 1xLysepuffer, resuspended the pellet by pipetting up and down and put it for 15 min at -80°. Then I thawed the cells, vortexed it, centrifuged again and transfered the supernatant to a fresh eppi. Then I put the supernatant at -20°.
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