Team:Stockholm/8 October 2010

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Latest revision as of 11:08, 26 October 2010

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	?	Incubate in RT, darkness for 10min Measure A₅₄₀ negative control ^{= without NADH}
NBT	1mg
phenazine methosulphate	10µg
EDTA	1µM
gelatin	1mg
phosphate	0.1M
NADH 1mM	0.1ml
tot	3ml
pH=7.8

The Faculty of Science at Stockholm University

Swedish Vitiligo association (Svenska Vitiligoförbundet)

@@ Line 1: / Line 1: @@
 {{Stockholm/Top2}}
 ==Andreas==
+[[image:Birthdaycake.jpg|Birthday cake]]
+'''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.
+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 &deg;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:
+ul * 2 = 1260 ul lysis buffer
+Wash buffer:
+X lysis buffer  12.6 ml
+Elution buffer:
+X lysis buffer 6.3 ml
+*PMSF
+* volume = 1260 * 1
+.6 ul PMSF added in lysis buffer
+*Imidazole
+* volume = 1260 * 10*10^-3
+.3 ul imidazole added in lysis buffer
+*Imidazole
+* volume = 1260 * 20*10^-3
+ul imidazole added in wash buffer
+*Lysozyme
+The tip of a spoon was added in lysis buffer
+*DNase
+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
+[[Image:SOD_activity.png‎|left|]]
+*Generates O<sub>2</sub><sup>-</sup>
+**Xanthine + xanthine oxidase
+**NADH + phenazine methosulphate
+**riboflavin + methionine
+*Reacts with O<sub>2</sub><sup>-</sup> 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 O<sub>2</sub><sup>-</sup>
+{|
+! mix
+|
+|
+|-
+| graded levels of SOD
+| ?
+| rowspan="9" |
+*Incubate in RT, darkness for 10min
+*Measure A<sub>540</sub>
+*negative control <sup>= without NADH</sup>
+|-
+| NBT
+| 1mg
+|-
+| phenazine methosulphate
+| 10µg
+|-
+| EDTA
+| 1µM
+|-
+| gelatin
+| 1mg
+|-
+| phosphate
+| 0.1M
+|-
+| NADH 1mM
+| 0.1ml
+|-
+| align="right" | tot
+| 3ml
+|-
+| pH=7.8
+|}
-Experiment canceled after about an hour; will be repeated next week.
+{{Stockholm/Footer}}