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Guidelines for using NEBuilder® HiFi DNA Assembly

NEBuilder HiFi DNA Assembly is a robust and powerful tool that can be used to combine different pieces of DNA. This chart provides recommendations, such as ratios of insert to vector as well as incubation times, for various scenarios. These guidelines may need to be adjusted to suit your particular situation.

Inserts
 Vector Recommended Ratio (insert : vector)
Amount setup in 20 µl (insert : vector (in fmol)) 		Recommended Time for Assembly Cloning Application Recommendations
3 kb insert

 

 Linear 2.1 kb plasmid 2 : 1
20 : 10		 15-60 min
  1. Conventional DNA assembly with restriction enzyme-digested fragments

  2. Typical DNA fragments from chemical synthesis or PCR
120 bp < insert < 200 bp

 Linear 5.4 kb plasmid 10-5 : 1
200-100 : 20		 15-60 min
  1. Typical DNA fragments from chemical synthesis or PCR

750 bp x 4, 20-30 bp overlap

 Circular assembly 1 : 1 : 1 : 1
20 : 20 : 20 : 20		 15-60 min
  1. Typical vector construction using DNA fragment from chemical synthesis or PCR

  2. Much higher DNA assembled efficiency than 15 bp overlap
750 bp x 4, 15 bp overlap

 Circular assembly 1 : 1 : 1 : 1
40 : 40 : 40 : 40		 15-60 min
  1. Typical vector construction using DNA fragment from chemical synthesis or PCR

  2. Conventional DNA assembly with restriction enzyme-digested fragments

  3. Typical DNA fragments from chemical synthesis or PCR
1,000 bp x 3, 25 bp overlap

 Linear 3.3 kb plasmid 1 : 1 : 1 : 1
50 : 50 : 50 : 50		 60 min
  1. Special DNA assembly with barcoded DNA fragments enriched from oligo chip pools
6 kb x 2 insert

 Linear 7 kb plasmid 1 : 1 : 1
25 : 25 : 25 		60 min
  1. Conventional DNA assembly with restriction enzyme-digested fragments

  2. Typical DNA fragments from chemical synthesis or PCR
70-mer ssDNA

 Linear 9.8 kb plasmid 200-50 : 1
1000-500 : 10		 15-60 min
  1. Make sgRNA Construct

  2. Insert short DNA sequence (1-25 bases)

  3. Construct DNA library
60-mer x 2

 Linear 2.6 kb plasmid 40-20 (annealed oligos) : 1
1000-500 : 25		 15-60 min
  1. Insert DNA with hairpin sequence

  2. Construct library

  3. Insert short DNA Sequences (15-30 bases)
60-mer x 5

 Linear 2.6 kb plasmid 40-20 (annealed oligos) : 1
1000-500 : 25		 15-60 min
  1. Construct library

  2. Insert short DNA sequence (<200 bp)
1000 bp x 5, 80 bp overlap

 Linear 3.3 kb plasmid 1 : 1 : 1 : 1 : 1 : 1
50 : 50 : 50 : 50 : 50 : 50		 60 min
  1. Conventional DNA assembly with restriction enzyme-digested fragments
450 bp x 11, 25 bp overlap

 Linear 3.3 kb plasmid 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1
50 (each insert) : 50		 60 min
  1. Typical vector construction using DNA fragment from chemical synthesis or PCR