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Plasmid & Protein Purification Database -Purification-

Species  
Locus tag ST1625
Products  
Plasmid ID R0460
Vector  
Purification Lot ST1625
Purified Date 2003.7.29
Protein conc. (mg/ml) 7.3
Total volume (ml) 0.7
Total protein (mg) 5.1

Culture Lot:

date host cell scale label weight of bacteira (g)
        16.65

Cell Lysis and Heat Treatment:

16.65 g of E. coli.
↓
30ml buffer/10ul B-ME(pH8.0)
↓
Sonication, output 8, 1 min on/1 min off, 5 times.
↓
Addition of 25ml buffer at 85 degC.
↓
Heat 20min, 85 degC.
↓
Incubation on ice for 10min.
↓
Centrifuge at 100,000 rcf for 2hr
↓
50 ml (45 ml of supernatant and 5 ml buffer).
 

Purification Procedure:

Step Column Buffer Gradient 1 Gradient 2 FlowRate (ml/min) Eluted
1 Resource Iso 6ml 20mM Tris-HCl(pH8.0),5mM B-ME,1.2M AS 1.2M AS 0M AS 4 between 1.2M AS to 1.0M AS
2 ToyoScreen PPG 600M 5ml 20mM Tris-HCl(pH8.0),5mM B-ME,1.5M AS 1.5M AS 0M AS 5 1.1M AS
3 Resource Q 6 ml 20mM Tris-HCl(pH8.0),5mM B-ME 0M NaCl 1M NaCl 4 0M NaCl
4 Bio-Scale CHT 10I 10mM KPi(pH7.0),5mM B-ME 10mM KPi 250mM KPi 3 between 25mM KPi and 50mM KPi
5 Superdex200HR10/30 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     0.5  
6            
7            
8            
9            

Comment:

Products were confirmed by MS.

2011.10.20

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