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General

We recommend selecting at least one restriction enzyme that will leave a sticky end to force the orientation of the insert.

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.

Dephosphorylating the vector to decrease background can be achieved with:

If you need to create blunt phosphorylated DNA ends (“polishing” the ends), you can use:

  • DNA End Repair Mix or T4 DNA polymerase or Klenow Fragment (large fragment) of E. coli DNA polymerase to generate blunt ends due to their 5’ →3’ DNA polymerase activity (filling-in of 5’ overhangs) and 3’→5’ exonuclease activity (chewing back of 3’ overhangs).

Digestion

In general, 1 unit of enzyme can digest 1 microgram of lambda DNA in 50 microliter solution in 1 hour at 37 degrees C. Please refer to the manual for specific temperature/time for your enzyme.

If you are able to find a buffer in which all 3 enzymes have sufficient activity (usually not lower than 50%), you can set up a single digestion with all 3 enzymes. It is important that the total volume of enzymes you add to your reaction is not more than 1/10th of the total reaction volume. The reason for this is that some enzymes have star activity if the concentration of glycerol exceeds 5%. If you are not able to find a buffer in which all your enzymes have sufficient activity, you will have to perform sequential digestions of the plasmid with the individual enzymes.

Ligation

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

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).
dATP is a competitive inhibitor of T4 DNA ligase. Phosphate will reduce ligation efficiency. Detergents in your ligation buffer will likely not affect activity. High levels (0.2 M) of Na2+, K+, Cs+, Li+, and NH4+ inhibit the enzyme almost completely. Polyamines, spermine, and spermidine also serve as inhibitors.

We offer T4 DNA Ligase (at 1 unit/μL or 5 units/μL) and ExpressLink™ T4 DNA Ligase.

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.

ExpressLink™ T4 DNA Ligase is a T4 DNA ligase that catalyzes the ligation of blunt or cohesive-end DNA fragments in only 5 minutes at room temperatures or ligation of PCR fragments with A’ overhangs in 15 minutes. ExpressLink™ T4 DNA Ligase undergoes superior exonuclease quality control assays for exonuclease-free ligations. The enzyme is supplied at a concentration of 5 units/μL.

You may have to try different ratios from 1:1 to 15:1 insert:vector.

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