A-Tailing of PCR products
This protocol is for adding a 3' terminal 'A' overhang onto your PCR products that were amplified using a blunt-ended, proof-reading enzyme (i.e Iproof). The product of this protocol is suitable for PCR-based cloning into vectors such as TA topo and pGEM T-easy.
- You must gel-purify or PCR purify the PCR product to remove the proof-reading enzyme from the reaction.
- Resuspend the gel-extracted product in 30 ul of EB or water.
- To a new tube, add:
17 ul of gel extracted product
2 ul 10X taq PCR buffer
0.5 ul dATP (100 uM)
0.5 ul Taq poylmerase
- Incubate the reaction at 72 for 20 - 30 minutes.
- Briefly spin down the reaction before ligation.
- Ligate 1.2 ul of this reaction into a 4 ul pGEM reaction.