Protocol for Bridging double-stranded DNA with a single-stranded DNA oligo using NEBuilder HiFi DNA Assembly (NEB #E2621)


This method allows you to assemble a single-stranded DNA (ssDNA) oligo insert and  a double-stranded DNA (dsDNA) vector fragment. The resulting construct can be used to prepare a library of inserts using the same overlap sequences at each end and a variable region in the middle, as in the following application note: Bridging dsDNA with a ssDNA Oligo and NEBuilder HiFi DNA Assembly to create an sgRNA-Cas9 Expression Vector

This protocol is recommended for the assembly of the following types of DNA fragment:

  • Short ssDNA inserts with 25 nucleotide (nt) overlaps
  • dsDNA vector linearized by PCR or restriction digest

The 3’ mismatch removal capabilities of the NEBuilder HiFi DNA Assembly allow for short barcodes (<10 nt) and residual bases from restriction digestions to be removed during assembly. Please see the following video for more information: NEBuilder® HiFi DNA Assembly Removal of 3´ end Mismatches


Single-stranded DNA oligo design

The ssDNA oligo should have 25-30 nucleotide overlaps on each end with the vector; there is no need to phosphorylate the 5’ end. This protocol is recommended for short insert sizes (1-25 bases). Please be aware, longer ssDNA oligos are more prone to secondary structure formation which may impact assembly performance.

DNA Quantities

This protocol uses a 1:200 (vector:insert) molar ratio with 0.005 picomoles of vector and 1 picomole of ssDNA oligo.


  1. Dilute resuspended oligos (100 μM stock) to a final concentration of 0.2 μM (0.2 pmol/μl) using TE buffer (10 mM Tris, 0.1 mM EDTA; pH 8.0) supplemented with 50 mM NaCl (or 1X NEBuffer r2.1). This can be done by taking 1 µl of 100 μM oligo stock and diluting it with 499 μl of buffer.
  2. Set-up the following reaction on ice:
  3. Component 20 µl Reaction Final Conc. Or Amount
    ssDNA Oligo
    (0.2 pmol/μl)*
    5 μl 1 pmol
    (1:200 vector:insert ratio)
    Vector (0.005 pmol/μl)* 1 μl 0.005 pmol
    Nuclease-free Water 4 μl  
    NEBuilder HiFi DNA Assembly Master Mix 2X 10 μl 1X

    * NEBioCalculator ( can help with DNA mass to molar quantity conversions for both ssDNA and dsDNA.

  4. Incubate the reaction at 50°C in a thermocycler for 60 min.  Transform 2 μl of assembled mix into NEB 5-alpha Competent E. coli (High Efficiency) (NEB #C2987) following the recommended protocol.

Note: If you are working with large plasmids >10 kb in size we recommend NEB® 10-beta Competent E. coli (High Efficiency) (NEB #C3019H). If your plasmid or insert contain repetitive sequences, we recommend NEB® Stable Competent E. coli (High Efficiency) (NEB #C3040H).