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Fast-Link DNA Ligation Kits
Ligation of Insert DNAs with Cohesive or Blunt Ends into Plasmid Vectors with Like Ends:
1. Assemble the folowing reaction in a microcentrifuge tube at room temperature, being
sure to add the ligase last:
a. Ligations of Insert DNA with Cohesive Ends
i. 1.5 ul 10X Fast-Link Ligation Buffer
ii. 1.5 ul 10 mM ATP
iii. x ul vector DNA (1 mole)
iv. x ul insert DNA (2 moles)
v. x ul sterile water to a volume of 14 ul
vi. 1 ul Fast-Link DNA Ligase
b. Ligations of Insert DNA with Blunt Ends
i. 1.5 ul 10X Fast-Link Ligation Buffer
ii. 0.75 ul 10 mM ATP
iii. x ul vector DNA (1 mole)
iv. x ul insert DNA (5 moles)
v. x ul sterile water to a volume of 14 ul
vi. 1 ul Fast-Link DNA Ligase
2. Incubate the reaction 5 minutes at room temperature for cohesive ends or 15 minutes for
blunt ends
3. Transfer the reaction to 70oC for 15 minutes to inactivate the Fast-Link DNA ligase
4. Spin briefly in a microcentrifuge.
5. Transform competent E. coli cells with 1/10 of the ligation reaction, keeping the volume
of the ligation to no more than 5% of the volume of competent cells.
6. If electroporating the ligated molecules, use no more than 2 ul of the ligation reaction
with 50 ul of electrocompetent cells.
7. Run 5 ul of the ligation reaction on an agarose gel
8. Visualize to determine the extent of ligation
Ligation of PCR Products with 3’ A-overhang into Plasmids with a 3’ T-overhang (T-vectors):
1. Gel extract or PCR cleanup the PCR product
2. Prepare a T-vector suitable for cloning PCR products
3. Assemble the following reaction in a microcentrifuge tube at room temperature, being
sure to add the ligase last:
a. Ligations of Insert DNA with Cohesive Ends
i. 1.5 ul 10X Fast-Link Ligation Buffer
ii. 0.75 ul 10 mM ATP
iii. x ul T- vector DNA (1 mole)
iv. x ul PCR product (1 mole)
v. x ul sterile water to a volume of 14 ul
vi. 1 ul Fast-Link DNA Ligase
4. Incubate the reaction for 1 hour at 16oC.
5. Transfer the reaction to 70oC for 15 minutes to inactivate the Fast-Link DNA ligase
6. Spin briefly in a microcentrifuge.
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7. Transform competent E. coli cells with 1/10 of the ligation reaction, keeping the volume
of the ligation to no more than 5% of the volume of competent cells.
8. If electroporating the ligated molecules, use no more than 2 ul of the ligation reaction
with 50 ul of electrocompetent cells.
9. Run 5 ul of the ligation reaction on an agarose gel
10. Visualize to determine the extent of ligation