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Plasmid & Protein Purification Database -Purification-

Species  
Locus tag ST0364
Products  
Plasmid ID 0576
Vector  
Purification Lot ST0364_2
Purified Date 2005.3.3-3.14
Protein conc. (mg/ml)  
Total volume (ml)  
Total protein (mg)  

Culture Lot:

date host cell scale label weight of bacteira (g)
        38

Cell Lysis and Heat Treatment:

38 g of E. coli.
200 ml of 20mM Tris-HCl (pH8.0), 5mM B-ME, 50mM NaCl.
Sonication, output 8, duty 50/3min, 4 times.
Centrifuge at 100,000 rcf for 30 min to obtain LS.
Heat 15 min 70 deg C.
Centrifuge at 100,000 rcf for 30 min.
 
 
 

Purification Procedure:

Step Column Buffer Gradient 1 Gradient 2 FlowRate (ml/min) Eluted
1 Resource Phe 6ml 20mM CAPS(pH9.5),5mM B-ME0.7M AS 0.7M AS 0M AS 5 from 0.56M AS to 0.24M AS
2 BioAssist Q 10x10 20mM CAPS(pH9.5),5mM B-ME 0M NaCl 0.3M NaCl 2.5 from 0.02M NaCl to 0.09M NaCl
3 BioAssist S 10x10 50mM MES(pH6.0),5mM B-ME 0M NaCl 0.5M NaCl 2.5 from 0.23M NaCl to 0.41M NaCl
4 HiLoad 16/60 Superdex200 50mM CAPS(pH9.5),5mM B-ME,150mM NaCl     1 90ml
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Comment:

 

2011.10.20

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