Team:Stockholm/8 October 2010
From 2010.igem.org
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Latest revision as of 11:08, 26 October 2010
Contents |
Andreas
Short day in lab - birthday celebrations!
Growth curve assay
Inoculated fresh LB with 0.3 mM IPTG with ON cultures to a startup OD of 0.2 and a total volume of 50 ml in 150 ml side-arm E-flasks.
Was advised to use a smaller cultures for the flask size. Experiment canceled after about an hour; will be repeated next week.
Removal of insertion in BioBrick suffixes
Plasmid prep
Spun down pSB1C3.SOD ON culture at 13,000 x g, 10 min. Supernatant discarded and cell pellet stored in -20 °C for later plasmid prep.
Nina
Protein purification
I performed a protein purification of SOD.His (N terminal). The pellet came from an IPTG induced 12 ml overnight culture.
Lysis buffer:
630 ul * 2 = 1260 ul lysis buffer
Wash buffer:
10X lysis buffer 12.6 ml
Elution buffer:
5X lysis buffer 6.3 ml
- PMSF
100 * volume = 1260 * 1
12.6 ul PMSF added in lysis buffer
- Imidazole
2 * volume = 1260 * 10*10^-3
6.3 ul imidazole added in lysis buffer
- Imidazole
2 * volume = 1260 * 20*10^-3
126 ul imidazole added in wash buffer
- Lysozyme
The tip of a spoon was added in lysis buffer
- DNase
20 ug/ml was added in lysis buffer
column equilibration
The work is carried out in a cold room.
- Ni-resin was vortexed and 1 ml was added in a drop column.
- 5 ml wash buffer was added and run through the column (to equilibrate the Ni-resin).
- The lysis mixed with the bacteria pellet was added in the column. However, the mixture seemed to plug the column and nothing could run through.
I left this in the cold room over the weekend to check with anyone at the department what to do and if I still can work with this material.
Mimmi
SOD activity assay
- Searching "web of science" for SOD activity assays
- Generates O2-
- Xanthine + xanthine oxidase
- NADH + phenazine methosulphate
- riboflavin + methionine
- Reacts with O2- and can be measured
- cytochrome C
- nitro-blue tetrazodium NBT
- adrenaline
- pyrogallol
- hydroxylamine
- Catalase-like test?
- No, since E. coli are catalase positive and SOD differences will not be detectable
- SOD activity test that chemically produces O2-
mix | ||
---|---|---|
graded levels of SOD | ? |
|
NBT | 1mg | |
phenazine methosulphate | 10µg | |
EDTA | 1µM | |
gelatin | 1mg | |
phosphate | 0.1M | |
NADH 1mM | 0.1ml | |
tot | 3ml | |
pH=7.8 |
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