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Team:Heidelberg/Notebook/Visualization/Notebook/1stweek
From 2008.igem.org
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.
- 11tg a plasmid with Amp-resistance which should allow the expression of GFP.
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
