Team:UNIPV-Pavia/Protocols/Ligation

From 2008.igem.org

(Difference between revisions)
 
Line 35: Line 35:
*'''Roche T4 Ligase'''
*'''Roche T4 Ligase'''
-
*'''Roche T4 Ligase Buffer'''
+
*'''10X Roche T4 Ligase Buffer'''
*'''ddH2O'''
*'''ddH2O'''
<br>
<br>

Latest revision as of 23:06, 1 October 2008

Home.jpg Home Unipv logo.jpg The Team And.jpg The Project Safety.jpg Biological Safety Dna.png Parts Submitted to the Registry
Laptop.jpg Dry Lab Pipette.jpg Wet Lab Math.gif Modeling Note.jpg Protocols Notebook.gif Activity Notebook


The protocols we used


Ligation

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

Materials needed:

  • Roche T4 Ligase
  • 10X Roche T4 Ligase Buffer
  • ddH2O


  • (For every ligation)
  • Add 50 ng of vector
  • Add Pv formula lig.png
  • Heat DNA mix at 65°C for 5 min for DNA denaturation
  • Add 1 µl of T4 Ligase buffer
  • Add 1 µl of T4 Ligase
  • 10 µl final volume
  • Incubate at 16°C overnight


  • 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.