Team:Heidelberg/Notebook/Visualization/Notebook/1stweek

  

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Overview

=First week=

Monday 01/09/2008

 * Preparation of TB media with an Agar concentration of 0.4 %, 0.2 % and 0.1 %.

First microscope test using MG1655 in 6 small chambers from Labtek:

3 types each type twice: I - 0.1% agar in LB II - 0.25% agar in LB III - 0.5% agar in LB

View:

I II III I II III


 * In I and II each chamber was filled with just 100 µl LB agar to cover the whole ground.


 * Because the III. solution was more viscous 150 µl were necessary to cover the ground.


 * The bacteria were put into one corner of a chamber. (2 µl of a MG1655 overnight culture) They have been set under the media or in between.


 * About that another 100 µl of a LB solution with 2 % agar were poured. After the second layer has solidified further 250 µl LB-media were added.


 * The chambers were incubated overnight at 37 °C. For microscope on the next day.

Notes: Because of surface tensions the solutions tend to be thicker at the borders of the chamber than in the center. -> In the further future 150 - 200 µl will be used.

Tuesday 02/09/2008
First approach in microscope seems to be good and can be followed up.

Yesterday overnight cultures of E.Coli HCB33 and E.Coli MG1655 have been inoculated in 3 ml LB media. Today they were diluted with additional 4 ml LB and splitted into two 3 ml falcon-samples. Followed by incubation at 37 °C.

Glycerol-stocks

 * The other 1 ml of each sample was used to create a glycerol stock.
 * 1 ml of the ONC mixed with 150 µl of 80% glycerol by vortexing. Storage at -80 °C after 30 minutes of rest.

First test producing fluorescent strains HCB33 and MG1655 strains.


 * Therefore our strains will be transformed with
 * 11tg a plasmid with Amp-resistance which should allow the expression of GFP.
 * BBa_I7100 from the registry, this should be expressing GFP, the production may be repressed with TetR. Kan-resistance.

Altogether there were 4 transformations. BBa_I7100 and 11tg each with HCB33 and MG1655.

The Transfomations was prepared using the following protocol:

Transformation

 * 1. -80 °C cultures thawing on ice.


 * 2. The spot has been cut from the registry folder and was transfered it to a 1,5 ml tube.


 * 3. 10 µl of prewarmed EB-Buffer have been put on the spot. Then tube was incubated at 37 °C for 20 minutes.


 * 4. 2-4 µl of plasmid DNA were given to 50 µl of competent cells and incubated for 20 minutes on ice.


 * 5. Afterwards the cells were heat shocked for about 45 seconds at 42 °C.


 * 6. Then each tube was put back into ice for 2 minutes.


 * 7. 400 µl of prewarmed LB has been added to each tube.


 * 8. Then the cells were incubated for 1 hour at 37 °C and 300 rpm.


 * 9. 150 µl and 300 µl of each tube were distributed on separate agar-plates containing the right antibiotics.


 * cells with 11tg on Amp plates
 * cells with BBa_I7100 on Kan_plates


 * 10. Plates were incubated overnight at 37 °C

Furthermore TB media, which contains 2% agar was produced and prepared for the4 autoclave.

The agar stocks prepared on monday were aliquotted on 1,5 ml eppi tubes. 1 ml per eppi.

Preparation of m9 media containing agar 2, 0.4, 0.2 and 0.1 % agar.

Wednesday 03/09/2008
Transformation from tuesday successful with most strains. Not successful with BBa_I7100 and MG1655. This will be repeated under slighly changed conditions:


 * spot was

Sunday 07/09/2008
back up

Overview

2nd Week