Latest revision as of 23:06, 1 October 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 22: / Line 22: @@
 *[[Team:UNIPV-Pavia/Protocols/Digestion|BioBrick digestion with restriction enzymes]]
 *[[Team:UNIPV-Pavia/Protocols/GelExtraction|DNA gel extraction]]
+*[[Team:UNIPV-Pavia/Protocols/Precipitation|DNA precipitation with sodium acetate]]
 *[[Team:UNIPV-Pavia/Protocols/AntarcticPhosphatase|Antarctic Phosphatase]]
 *[[Team:UNIPV-Pavia/Protocols/Ligation|Ligation]]
@@ Line 28: / Line 29: @@
 <h1>Ligation</h1>
-''(estimated time: 3 hours and 30 min)''
+''(estimated time: 20 min + 12-16 hours overnight incubation)''
 <br>
 <br>
 '''Materials needed:'''
-*'''MgCl2'''
+*'''Roche T4 Ligase'''
-*'''Buffer'''
+*'''10X 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
+*Add 50 ng of vector
-**0.6 µl MgCl2
+*Add [[Image:pv_formula_lig.png]]
-**0.4 µl dNTPs
+*Heat DNA mix at 65°C for 5 min for DNA denaturation
-*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
+*10 µl final volume
+*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>