TA subcloning using Promega pGEM-T Easy Vector System

(Promega, Cat#: A1360).

DNA purification for subcloning (Promega Cat#: A7170, A2180).

Mix 10 ul of Langdon amplicon with 50 ul of Direct Purification buffer and add 1 ml of resin. Mix by vortexing. Assemble purification Minicolumns and transfer resin/DNA mix into the column, apply vacuum. Wash the column with 2 ml of 80% isopropanol. Centrifuge the minicolumn at 10,000 x g for 2 min to remove the residual isopropanol. Apply 50 ul of water to the minicolumn and wait 1 min. Centrifuge the minicolumn for 20 sec at 10,000 x g to elute DNA fragment. (For details see kit’s manual).

Ligation reaction:

3.0 ul purified PCR product

5.0 ul 2X ligation buffer (supplied with TA cloning kit)

0.2 ul pGEM-T Easy vector (10ng)

1.0 ul T4 DNA ligase (Promega, supplied with the kit)

0.8 ul water

Overnight ligation at +4˚C.