Team:Stockholm/11 October 2010
From 2010.igem.org
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+ | =Johan= | ||
+ | ==Ligation== | ||
+ | his-bFGF and bFGF-his into gel purified pEX, both insert and vector cut with XbaI and PstI | ||
+ | |||
+ | 7 µl insert, 0,5 µl pEX | ||
+ | |||
+ | 1 µl ligase T4 | ||
+ | |||
+ | 2 µl 10x buffer | ||
+ | |||
+ | 9,5 µl H2O | ||
+ | |||
+ | 1 h 37 °C | ||
+ | |||
+ | ==Transformation== | ||
+ | |||
+ | 3 µl of both pEX + his-bFGF and pEX + bFGF-his into top10 cells | ||
+ | |||
+ | ==Overnight culture== | ||
+ | Made overnight culture from 10 colonies of tat-bFGF-his which didnt seem to be cut last time | ||
+ | |||
+ | {{Stockholm/Footer}} |
Latest revision as of 19:47, 27 October 2010
Contents |
Nina
Continuation of protein purification
I got help with the plugged drop column and got the fractions I needed of the lysis, wash and elution buffers.
These samples of 0.5 ml were stored in a freezer for running on an SDS gel.
Andreas
Removal of insertion in BioBrick suffixes
Plasmid prep
From 8/10 stored pellet
- 50 μl elution buffer.
DNA concentration | ||
---|---|---|
Sample | Conc [ng/μl] | A260/A280 |
pSB1C3.SOD | 163.1 | 1.94 |
Digestion
pSB1C3. SOD | |
---|---|
10X FastDigest buffer | 2 |
DNA (1 μg) | 12.3 |
dH2O | 3.7 |
FD EcoRI | 1 |
FD SpeI | 1 |
20 μl |
- Incubation: 37 °C, 30 min
Gel verification
1 % agarose, 140 V
Expected bands
- Vector: 2175 bp
- Insert: 495 bp
Results
Band slightly larger than expected, but this may also be due to bound restriction enzymes and/or other disturbances. Proceeded to gel extraction.
Gel extraction
Digested pSB1C3.SOD separated on 1 % agarose gel at 110 V, 17 μl sample volume. SOD band extracted and DNA, as well as DNA from samples stored in -20 °C 7/10, were purified using the E.Z.N.A. Gel Extraction kit.
DNA measurements extremely low, but proceeded to ligation/cloning anyway.
Ligation
pSB1C3. IgGp | pSB1C3. bGFG | pSB1C3. ProtA | pSB1C3. yCCS | pSB1C3. SOD | |
---|---|---|---|---|---|
10X T4 Ligase buffer | 2 | 2 | 2 | 2 | 2 |
Vector DNA | 3 | 3 | 3 | 3 | 3 |
Insert DNA | 14 | 14 | 14 | 14 | 14 |
dH2O | 0 | 0 | 0 | 0 | 0 |
T4 DNA ligase | 1 | 1 | 1 | 1 | 1 |
20 μl | 20 μl | 20 μl | 20 μl | 20 μl |
- Incubation: 22 °C, 15 min
Transformation
- Mod. quick transformation
- Top10
- 3 μl ligation mix
- 30 min incubation on ice
Mimmi
SOD activity assay
- Nondenaturing gel electrophoresis
- Staining with NBT
- SOD activity test with enzyme producing O2-
- mix 750µl culture with 600µl ice cold ethanol:chloroform (5:3)
- vortex 20s, incubate 3min, vortex again 20s
- centrifuge at 2500g for 45min in 4°C
- keep supernatant on ice
mix | |
---|---|
supernatant |
|
phosphate buffer 50mM | |
EDTA 5mM | |
bovine serum albumin 40mg/l | |
NBT 0.2g/l | |
xanthine + oxidase 0.1mM |
- SOD activity test with illumination
mix | ||
---|---|---|
riboflavin | 1.17*10-6M |
|
methionine | 0.01M | |
sodium cyanide | 2*10-5M | |
NBT | 5.6*10-5M | |
potassium phosphate | 0.05M | |
pH=7.8 | ||
SOD | 20-260ng/ml | |
tot 3.0ml |
Johan
Ligation
his-bFGF and bFGF-his into gel purified pEX, both insert and vector cut with XbaI and PstI
7 µl insert, 0,5 µl pEX
1 µl ligase T4
2 µl 10x buffer
9,5 µl H2O
1 h 37 °C
Transformation
3 µl of both pEX + his-bFGF and pEX + bFGF-his into top10 cells
Overnight culture
Made overnight culture from 10 colonies of tat-bFGF-his which didnt seem to be cut last time
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