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

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Notes: Because of surface tensions the solutions tend to be thicker at the borders of the chamber than in the center.<br>
Notes: Because of surface tensions the solutions tend to be thicker at the borders of the chamber than in the center.<br>
-> In the further future 150 - 200 µl will be used.
-> In the further future 150 - 200 µl will be used.
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==Tuesday 02/09/2008==
==Tuesday 02/09/2008==
First approach in microscope seems to be good and can be followed up.
First approach in microscope seems to be good and can be followed up.
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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.<br>
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.<br>
Followed by incubation at 37 °C.
Followed by incubation at 37 °C.
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===Glycerol-stocks===
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* The other 1 ml of each sample was used to create a glycerol stock.<br>
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** 1 ml of the ONC mixed with 150 µl of 80% glycerol by vortexing. Storage at -80 °C after 30 minutes of rest.
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First test producing fluorescent strains HCB33 and MG1655 strains.
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*Therefore our strains will be transformed with<br>
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**11tg a plasmid with Amp-resistance which should allow the expression of GFP.<br>
 +
**BBa_I7100 from the registry, this should be expressing GFP, the production may be repressed with TetR. Kan-resistance.
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 +
Altogether there were 4 transformations.<br>
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BBa_I7100 and 11tg each with HCB33 and MG1655.
 +
 +
The Transfomations was prepared using the following protocol:
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 +
===Transformation===
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* 1. -80 °C cultures thawing on ice.
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 +
* 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.
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 +
* 5. Afterwards the cells were heat shocked for about 45 seconds at 42 °C.
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 +
* 6. Then each tube was put back into ice for 2 minutes.
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* 7. 400 µl of prewarmed LB has been added to each tube.
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 +
* 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==
==Wednesday 03/09/2008==
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Transformation from tuesday successful with most strains.<br>
 +
Not successful with BBa_I7100 and MG1655.<br>
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This will be repeated under slighly changed conditions:<br>
 +
 +
*spot was
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==Thursday 04/09/2008==
==Thursday 04/09/2008==
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==Friday 05/09/2008==
==Friday 05/09/2008==
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==Saturday 06/09/2008==
==Saturday 06/09/2008==
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==Sunday 07/09/2008==
==Sunday 07/09/2008==
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[https://2008.igem.org/Team:Heidelberg/Notebook/Visualization/Notebook/1stweek back up]
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[[Team:Heidelberg/Notebook/Visualization/Notebook/|Overview]]
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[[Team:Heidelberg/Notebook/Visualization/Notebook/2ndweek|2ndWeek]]
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[[Team:Heidelberg/Notebook/Visualization/Notebook/2ndweek|2nd Week]]

Latest revision as of 14:11, 29 October 2008

Overview

Contents

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

Thursday 04/09/2008

Friday 05/09/2008

Saturday 06/09/2008

Sunday 07/09/2008

back up

Overview

2nd Week

Retrieved from "http://2008.igem.org/Team:Heidelberg/Notebook/Visualization/Notebook/1stweek"