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Team:Groningen/Notebook/Peter
From 2010.igem.org
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| + | Expression experiments: | ||
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| + | Numero dos | ||
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| + | 1 growing: Thursday 26/08/2010 | ||
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| + | 2: analasis friday 27/08 (supernatant preperation - 4: ne1t=4, 1,1t=4, tasat=2, deltat=2)) and sunday, 29/08/2010, 01/09/2010: rest supernatant | ||
Latest revision as of 12:48, 1 September 2010
Bascillus Subtilus, easy info:
http://www.mobitec.de/int/products/bio/04_vector_sys/index.php?bac_sub.html
http://subtiwiki.uni-goettingen.de/wiki/index.php/SubtiPathways
http://subtiwiki.uni-goettingen.de/wiki/index.php/Main_Page
09/08
Cell wall isolation according to protocol: Cell disruption, samples are frozen.
Culture Bascillus over night, all four strains: Rok, DegU, SigF, Sp0A
10/08
Cell wall isolation according to protocol: Cell disruption again, continue according to protocol
Dry-freeze over night
Culture Bascillus over a extra night, all four strains: Rok, DegU, SigF, Sp0A
11/08
Chaplin isolation:
4x approximately 15 mg dry cell wall (180 ul TFA),
4x approximately 10 mg dry cell wall (100 ul TFA),
8x approximately 5 mg dry cell wall (100 ul TFA),
Cultured bascillus strains are taken out of the stove, medium will be used for degredation experiment.
Pellicle: ROK + SigF
DEGREDATION EXPERIMENT:
3x appr. 5 mg samples + ROK, DegU, SigF medium (+ 1 control normal medium)
3x appr. 5 mg samples + ROK, DegU, SigF lysed medium (+ 1 control lysis normal medium)
3x appr. 15 mg samples + ROK, DegU, SigF medium (+ 1 control normal medium)
3x appr. 10 mg samples + ROK, DegU, SigF medium (+ 1control normal medium)
normal medium is with 2% glucose for lysis
normal medium is TY for the rest
12.45: lysis at 37 degrees for 1 hour, 20 mg/ml lysozyme (control also)
13.45: incubation with bacillus and control media
12-08-2010
Hydrophobin samples were prepared for sds-gel at 14.00
Lysis samples were spinned off (13000 rpm/4 min), the samples were not boiled, the samples were prepared in 05,x SDS loading buffer
25 uL of sample was loaded on the SDS gel in the following order:
on gel 1:
5ulmarker-15ctrl-15sigf-15degu-15rok-rokl-sigfl-degu-l-ctrll-10ulmarker
on gel 2:
5ulmarker-10ctrl-10sigf-10degu-10rok-5ctrl-5sigf-5degu-5rok-10ulmarker
The 1 mm thick gel 1 was prepared using a 0,75 mm comb: Might be bad
Gel was runned on 60 v for 15 min
Then gel was runned on 120 v
Expression experiments:
Numero dos
1 growing: Thursday 26/08/2010
2: analasis friday 27/08 (supernatant preperation - 4: ne1t=4, 1,1t=4, tasat=2, deltat=2)) and sunday, 29/08/2010, 01/09/2010: rest supernatant
