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 3 : 1
30 : 10
15-60 min
  1. Conventional DNA assembly with restriction enzyme-digested fragments

  2. Typical DNA fragments from chemical synthesis or PCR

75 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

Self-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

Self-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
27 : 27 : 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 100-50 : 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)

100 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