Revision as of 12:43, 1 July 2008

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The protocols we used

(estimated time: 20 min + 12-16 hours overnight incubation)

Materials needed:

Then, ligation can be conserved at 4°C or can be transformed
Before transformation you have to inactivate T4 Ligase:
- Heat ligation at 65°C for 10 min.

@@ Line 33: / Line 33: @@
 '''Materials needed:'''
-*'''MgCl2'''
+*'''Roche T4 Ligase'''
-*'''Buffer'''
+*'''Roche T4 Ligase Buffer'''
-*'''dNTPs'''
 *'''ddH2O'''
-*'''Taq Polymerase'''
-*'''VF2 primer'''
-*'''VR primer'''
 <br>
-*For every DNA sample you want to amplify, put:
+*(For every ligation)
-**2 µl buffer
+*Heat vector at 65°C for 5 min for DNA denaturation
-**0.6 µl MgCl2
+*Add 50 µg of vector
-**0.4 µl dNTPs
+*Add <math>6*50*length(insert)/length(vector)</math>
-*Now you can add a loading buffer to the solution and perform electrophoresis to check the amplified sequence length.
+*Add 1 µl of T4 Ligase buffer
+*Add 1 µl of T4 Ligase
+*Incubate at 16°C overnight
+<br>
+*Then, ligation can be conserved at 4°C or can be transformed
+*Before transformation you have to inactivate T4 Ligase:
+**Heat ligation at 65°C for 10 min.
 <br>