Bio-Rad Ligation and Transformation Module - Manual (Page 41)

Setting Up the Blunting Reaction
This reaction removes the 3' nucleotide overhang left by the Taq DNA
polymerase that would prevent blunt end ligation.
1. Label a microcentrifuge tube with your initials, the name of your amplified
gene, and "ligation."
2. Pulse spin the stock tubes containing the ligation reaction buffer and
proofreading polymerase in a microcentrifuge for 10 sec to force contents
to bottom of tubes prior to use.
Note: When pipetting very small volumes, take special care. When pulling
up reagents, make sure only the soft stop of the pipet is used even though
it may feel like a very small movement. Also, look at the end of the pipet tip
and make sure that the correct volume of reagent is in the tip. After adding
the reagent to the tube, be sure that the pipet tip is empty. Never reuse a
pipet tip.
3. Set up a blunting reaction with the following reagents:
Reagent Amount
2x ligation reaction buffer 5.0 µl
Purified PCR product 1.0 µl
Sterile water 2.5 µl
Proofreading polymerase 0.5 µl
Total 9 µl
Note: The amount of PCR product added to the reaction may be
increased to 2 µl if the PCR product is not very intense when analyzed on
an agarose gel — for example if it is not as intense as the 1 kb band in the
molecular weight marker. If the amount of PCR product is increased,
remember to decrease the volume of sterile water to 1.5 µl to compensate
and keep the total volume of 9 µl.
37