Hello everyone,

I have problem about one blunt end and one sticky end ligation. I'm trying to ligate insert size 2,017 bp and vector size 5,300 bp. The PCR product (Insertion, ~2,017 bp) has smaI and hindIII sites. PCR product was cloned into pGEM-t easy vector and then double digestion. Digestion of PCR product from pGEM-t vector was cloned into another vector (~5,300bp). Gel purification and solution purification were performed before ligation. I used transformation by heat shock at 42 degree celsius for 90s.

I'm digesting the vector (concentration~800 ng) and pGEM-insert (concentration~800 ng) with Fastdigest smaI (blunt end) and Fastdigest hindIII (sticky end) from Thermo scientific. The company recommend digestion protocol for 5-15 minutes, I increased the digestion to 3 hours. I performed double digestion at the same time with incubated at 37 degree celsius. This smaI-HF and hindIII-HF can digest at 37 degree celsius. I did vector dephosphorylation before ligation. After digestion, I purified the vector and insert by column and used blunt end protocol with PEG ligation from Thermo scientific. I used 10:1 insert:vector ratio (total 20 ul) and ligate at 4 degree celsius for 3 h and then at 22 for overnight. I used 5 ul of ligation mixture to transform. After transformation, I got 9 colonies and PCR showed negative amplification.

Competent cells are fine. SmaI and hindIII site on vector are quite far. When I did double digestion of pGEM-insertion, I got vector size and insert size correctly. I think double enzyme digestion is work.

Do you have any recommend or suggestion to solve this problem?

Thank you very much.