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--- purification:tev:s_protocol [2016/07/13 15:34] – bonnefon
+++ purification:tev:s_protocol [2023/11/01 20:19] (current) – external edit 127.0.0.1
@@ Line 2: / Line 2: @@
 **j-1**
-  * Transform BL21/LysS strain on KanR with optimized TEV plasmid (stockpBS2440).
+  * Transform BL21/LysS strain on KanR with optimized TEV plasmid (stock pBS2440).
 **j**
-  * Inoculate 25 ml medium A with one colony at the end of the afternoon.
+  * Inoculate 25 ml medium A((LB, Cm 25 μg/ml, Kan 10 μg/ml)) with one colony at the end of the afternoon.
   * Grow at 37°C.
@@ Line 18: / Line 18: @@
 **j+2**
-  * Resuspend pellet in Buffer 1.
+  * Resuspend pellet in Buffer 1((20 mM Tris-HCl pH 8.0, 10 mM imidazole, 200 mM Nacl, 2 mM β-ME, 0.2% NP40))
-  * Adjust to 20 ml with buffer 1.
+  * Adjust to 20 ml with Buffer 1.
   * Pass through French-Press 2 times.
   * Spin down with ss34 12,000 rpm for 30 min.
@@ Line 25: / Line 25: @@
   * Rotate 1h at 4°C.
   * Transfer in an Econo-column (Biorad).
-  * Wash with 15ml buffer 1.
+  * Wash with 15ml Buffer 1.
-  * Wash with 5 ml buffer 2.
+  * Wash with 5 ml Buffer 2((20 mM Tris-HCl pH 8.0, 10 mM imidazole, 1 M NaCl, 2 mM β-ME, 0.2% NP40))
-  * wash with 10 ml buffer 1.
+  * wash with 10 ml Buffer 1.
-  * Elute with 4x0.5 ml buffer 3 in 0.5 ml glycerol tube (50% glycerol final).
+  * Elute with 4x0.5 ml Buffer 3((20 mM Tris-HCl pH 8.0, 200 mM imidazole, 200 mM NaCl, 2 mM β-ME, 0.2% NP40)) in 0.5 ml glycerol tube (50% glycerol final).
   * store at -80°C.