August

From 2008.igem.org

_august

Aug. 8th 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%.

Aug. 11th 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)

Aug. 12th 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°

Aug. 13th 2008

There again were no colonies on the plates.

Preparing T-cells for MTT-assay (Normann) To test the Mg2+ tolerance of the T-cells 800µl medium was given to 100µl cellsuspension and 100µl MgCl2/MgAc Solution with different concentrations.

well --- Mg2+ [mM]
1 ------- control
2 ------- 12.5
3 ------- 8.75
4 ------- 6.25
5 ------- 5
6 ------- 3.75
7 ------- 2.5
8 ------- 1.25

Aug. 14th 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

Aug. 15th 2008

There were colonies on the plates from both parts.
Splitting T-cells(Normann)
The cells (15ml) were spun down at 1200 rpm (5min) and resolved with 10ml PBS. Then they were spun down again and resolved in 15ml fresh medium. 5ml was given to a dish and 15ml additional medium was added.
MTT-assay Normann

centrifuging 1ml cellsuspension at 1200 rpm (5min)-> discarding the supernatant
adding 200µl fresh medium and 50µl MTT
waiting for 4h
centrifuging again at 1200rpm (5min) -> discarding the supernantant
resolving the pellet with 400µl DMSO and adding 50µl Soerensen
incubation for 10 min

measuring at 570nm (autozero with DMSO+Soerensen)

Mg2+ [mM]	A (MgCl2)	A (MgAc)
12,5	0,388	0,284
8,75	0,488	0,23
6,25	0,568	0,217
5	0,328	0,22
3,75	0,365	0,311
2,5	0,255	0,295
1,25	0,207	0,241
control	0,449	0,395

Aug. 17th 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)

Aug. 18th 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°

Aug. 19th 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

Aug. 22nd 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

Aug. 25th 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.

Aug. 26th 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.

Aug. 28th 2008

Harvesting cells
For the ONPG Test we decided to use cells after several timepoints. So I harvested cells after 48h and 68h.
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°.
Control was done with untransfected cells using the same procedure.
Result