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Plasmid & Protein Purification Database -Purification-

Species  
Locus tag ST1790
Products  
Plasmid ID R0468
Vector  
Purification Lot ST1790_1
Purified Date 2003.7.29
Protein conc. (mg/ml) 1.5
Total volume (ml) 0.5
Total protein (mg) 0.75

Culture Lot:

date host cell scale label weight of bacteira (g)
        20.1

Cell Lysis and Heat Treatment:

20.14 g of E. coli.
35ml buffer(20mM Tris-HCl,,50mM NaCl),5mM B-ME(pH8.0)10ul
Sonication, output 8/duty 50, 1 min, on ice 1 min.
20ml buffer(85?),15min heat. Heat 20min, 85 deg C.
Centrifuge at 100,000 rcf for 2hr
70?,20min re-heat
Centrifuge at 5,000 rcf for 60min.
50 ml (42 ml supernatant and 8 ml buffer).
 

Purification Procedure:

Step Column Buffer Gradient 1 Gradient 2 FlowRate (ml/min) Eluted
1 ToyoScreen PPG 650M 20mM Tris-HCl(pH8.0),5mM B-ME,1.2M AS 1.2M AS 0M AS 5 Flow through
2 ToyoScreen PPG 650M 20mM Tris-HCl(pH8.0),5mM B-ME,1.5M AS 1.5M AS 0M AS 4 1.2M AS
3 Resource Q 6 ml 20mM Tris-HCl(pH8.0),5mM B-ME 0M NaCl 1M NaCl 4 Flow through
4 Bio-Scale CHT10I 10mM KPi(pH7.0),5mM B-ME 10mM KPi 2510mM KPi 4 150mM KPi
5 Superdex200HR10/30 20mM Tris-HCl(pH8.0),5mM B-ME,150mM NaCl     0.5 16ml/24ml
6            
7            
8            
9            

Comment:

Products were confirmed by Mass in entire sequence.

2011.10.20

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