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From 2010.igem.org

Contents 1 Nina 1.1 Continuation of protein purification 2 Andreas 2.1 Removal of insertion in BioBrick suffixes 2.1.1 Plasmid prep 2.1.2 Digestion 2.1.3 Gel verification 2.1.4 Gel extraction 2.1.5 Ligation 2.1.6 Transformation 3 Mimmi 3.1 SOD activity assay 4 Johan 4.1 Ligation 4.2 Transformation 4.3 Overnight culture

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	Measure A546 for 10min at 25°C xanthine oxidase activity ajusted to increase in A546 0.1 U/min
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	Illuminate so the absorbance increases with 0.142U/6min A560 linear increase with time
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