Current revision

PcTF Subcloning in E. coli

01/12/2013

Ligation of KAH111, KAH112 and C-His vector in DH5α-T and BL21

The 4 μL of C-His Vector from stock is diluted with 16 μL of nuclease free water, then cut with E/P and purified from the gel.
Ligations

Ligation	</font>
1. E. coli BL-21 KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng	KAH111/C-His 3:1 No Colonies
2. E. coli DH5α-T KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 26ng	KAH111/C-His 3:1 No Colonies
3. E. coli BL-21 KAH112(E/P)/size, 22.8 ng + C-His(E/P)/3600, 26ng	KAH112/C-His 3:1 No Colonies
4. E. coli DH5α-T KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng	KAH112/C-His 3:1 No Colonies
5. C-His(E/P)/ 26 ng (Control Plate)

Calculations are for the ng of insert we need to get a 3:1 ratio of insert molecules to 50 ng vector molecules

@@ Line 26: / Line 26: @@
 |}
+* Calculations are for the ng of insert we need to get a 3:1 ratio of insert molecules to 50 ng vector molecules
 {| {{table}} cellspacing="3" <!-- Ligation rxn table -->
 | &nbsp;             || 1    || 2  || 3   || 4 || 5
@@ Line 43: / Line 44: @@
 * The incubation time for the ligation process was 30min at room temperature.
+* Fast transformation, 30 min on ice.