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Latest revision as of 14:11, 29 October 2008

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

Thursday 04/09/2008

Friday 05/09/2008

Saturday 06/09/2008

Sunday 07/09/2008

back up