FAQ: How should fragments be prepared for assembly using NEBuilder HiFi?

Fragments can be prepared by the following methods:

  1. PCR-generated fragments can be cleaned-up by using Monarch PCR column or Exo-CIP Rapid PCR Cleanup Kit if amplicon purity is greater than 95%.  If plasmid DNA was used as template during PCR, it can be removed by DpnI treatment if necessary.  If multi-bands are observed, we recommend optimizing the PCR.  If this is not possible gel purification is recommended.  Gel extraction can introduce guanidine thiocynate (from the dissolving buffer) that can reduce the efficiency of the assembly reaction.  To minimize this contamination, trim the gel slice so that a smaller amount of gel dissolving buffer can be used.

  2. Restriction enzyme digestion of a plasmid can be performed followed by heat-inactivation or column purification.

  3. Commercially ordered fragments can be re-suspended in nuclease-free water or TE buffer and directly used in the assembly reaction.
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