• My NEB
  • Print
  • PDF
  • Ligation Protocol for NEB PCR Cloning Kit

    Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community.

    1. Assemble ligation reactions using the chart below as a guide. Mix the first 4 components before adding 5 μl of the cloning mix consisting of 4 μl Cloning Mix 1 and 1 μl Cloning Mix 2, for a total of 10 μl per ligation reaction. It is recommended that first-time users of this kit perform the positive control ligation reaction.

       Linearized pMiniT Vector (25 ng/ μl)  1 μl (25 ng) 1 μl (25 ng) 
       Insert*  1-4 μl*  -
       Amplicon Cloning Control (1 kb) (15 ng/ μl) -  2 μl (30 ng)
       H20  to 5 μl  2 μl
       Cloning Mix 1  4 μl  4 μl
       Cloning Mix 2  1 μl  1 μl
       Total Volume  10 μl  10 μl
      *For purified PCR amplicon products, the amount of insert to be added can be calculated by relative length or molar calculations. For illustrative purposes calculations are shown below; however, the NEBiocalculator web tool is a quick and convenient way to determine the insert amounts for all cloning reactions. Formulas below use the recommended values of 25 ng of linearized vector (2525 bp) per reaction and an insert-to-vector ratio of 3:1.

      1. Relative length calculations:
        ng insert to be added = (3)(25 ng vector) (bp of insert/2525 bp of vector)

      2. Molar calculations:
        1. Convert the 25 ng vector present in the ligation reaction to pmoles:
          (25 ng vector)(1000)/(650 daltons per base pair)(number of base pairs in vector or
          2525) = (25)(1000)/(650)(2525) = 25000/1641250 = 0.015 pmoles vector
        2. Calculate a 3-fold molar amount of insert to add to each ligation:
          (3)(0.015 pmoles vector) = 0.045 pmoles insert
        3. Convert the pmoles insert amount to ng insert to be added:
          ng insert to be added = (0.045 pmoles insert)(base pairs in insert)(650 daltons per
          base pair)/1000

      As examples, these calculations will yield insert levels of 15 ng (500 bp insert), 30 ng (1 kb insert) or 60 ng (2 kb insert).

      For unpurified PCR amplicons from reactions yielding a specific product, use 1 μl or less of the reaction as insert to approximate achieving a 3:1 molar ratio of insert:vector. For high yield PCRs, less than 1 μl may be required to avoid adding a vast excess of insert that would result in insert ligating to both ends of the linearized vector.  Do not use more than 1 μl, as carryover material from the PCR reactions can inhibit ligation or transformation. To estimate the concentration of PCR product for the above calculations, compare the reaction yield to known amounts of DNA fragments in a marker lane, such as our Quick-Load® Purple 2-Log DNA Ladder (NEB #N0550S).

    2. Incubate at room temperature (25°C) for 5–15 minutes. (While 5 minutes is sufficient, 15 minutes will increase transformation levels for amplicons with single base overhangs)

    3. Incubate on ice for 2 minutes.

    4. Transform immediately or store at -20°C. For best results, transform into NEB 10-beta Competent E. coli (NEB #C3019), which are supplied with NEB #E1202.

    NEB® PCR Cloning Kit
    NEB® PCR Cloning Kit (Without Competent Cells)