Ligation of PCR product into TOPO 2.1 vector

Before proceeding with ligation, please make sure that you have done a PCR clean-up reaction if necessary, following the Zymo DNA clean and concentrator kit.

If needed [only if PCR product has been sitting in the freezer for >5 days]:

Adding A’s To ensure the presence of 3’ A overhangs on the PCR product add 1 to 5 units of Taq polymerase (1ul of GoTaq) to your PCR tube. Place in PCR machine programmed to run 94oC for 3min and incubate the tube at 72°C for 10 mins.

Ligation Protocol:

1. Ensure all ligation reagents are thawed. Salt solution (clear cap) can be thawed at room temp and then placed on ice, but the TOPO vector (yellow cap) should be kept on ice at all times.

2. In a clearly-labeled PCR tube, create the following 6μl ligation mixture by adding the reagents in the order listed:

Reagent:

1 μl Water

1 μl Salt Solution (1.2M NaCl, 0.06M MgCl2)

3 μl PCR Product

1 μl TOPO Vector

3. Mix the reaction very gently with a pipette tip and incubate for 10 min at room temperature, NOT on ice. While sample is incubating, return the rest of your PCR product to the -20C freezer in case we need to run the ligation again.

4. Place the reaction on ice and proceed immediately with transformation, or store ligation at -20C. After setting up transformations KEEP UNUSED/ADDITIONAL LIGATION AT -20oC (don’t discard this tube we can come back to this step).