ADN ligase T4 (5 U/μl)

The main difference between the 2 enzymes is that E. coli DNA Ligase cannot ligate blunt dsDNA fragments. Both ligases can be used to repair single stranded nicks in duplex DNA and to perform cohesive or sticky end ligations. E. coli DNA Ligase is generally used to seal nicks during second strand cDNA synthesis, since T4 DNA Ligase could result in formation of chimeric inserts.

Please consider the following suggestions:
1– Try different molar ratios of insert to vector. Having an excess of insert is usually what will work, try 1:1 to 15:1 insert:vector.
2– Try increasing the time of the ligation at 37 degrees C.
3– Try performing the ligation at 16 degrees C overnight (you can set it up on your PCR machine).

Make sure you have inactivated the ligase and store the ligation reaction at 4 degrees C.

You can have all of the below controls or select the one you consider the most appropriate to the problem you are facing:
1– Transform the E. coli with circular plasmid to assess the competency of the cells (how well they are taking up DNA).
2– Transform and plate the dephosphorylated vector. It will help you assess how well the dephosphorylation worked and what proportion of colonies in your ligation transformation plate could be false positives (re-ligated vector or background).
3– Use T4 DNA igase to re-ligate your cut vector, or lambda DNA/Hind III marker. It will help you assess whether the ligase itself is working properly.

dATP is a competitive inhibitor. Phosphate will reduce ligation efficiency. Detergents in your ligation buffer will likely not affect activity. High levels (0.2M) Na2+, K+, Cs+, Li+, and NH4+ inhibit the enzyme almost completely. Polyamines, spermine, and spermidine also serve as inhibitors.