Bio-Rad Ligation and Transformation Module - Manual (Page 42)

4. Close the cap and mix well. Centrifuge in a microcentrifuge for 10 sec
to collect the contents at the bottom of the tube.
This step is essential due to the very small volume used in this reaction.
5. Place the tube at 70°C for 5 min.
6. Place tube on ice to cool for 2 min.
This recondenses water vapor to maintain reaction volume.
7. Once cool, centrifuge the tube briefly to collect the contents at the bottom
of the tube. Place tube at room temperature.
Setting Up the Ligation Reaction
This reaction inserts the PCR product into the pJet1.2 plasmid vector.
8. Briefly centrifuge the stock tubes containing the pJet1.2 blunted vector
and the T4 DNA ligase in a microcentrifuge to force the contents to
bottom of tubes prior to using.
9. Setup a ligation reaction with the following reagents:
Reagent Amount
Blunting reaction (already in 9.0 µl
microcentrifuge tube)
T4 DNA ligase 0.5 µl
pJet1.2 blunted vector 0.5 µl
Total 10 µl
10. Close the cap and mix well. Centrifuge briefly in a microcentrifuge to
collect the contents at the bottom of the tube.
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